Search

Introduction Tigecycline is a potential alternative to trimethoprim–sulfamethoxazole in treating Stenotrophomonas maltophilia infections due to its potent in vitro antimicrobial activity. Clinical evidence regarding the use of tigecycline in the treatment of S. maltophilia infections is scarce. In this study, we assessed the efficacy of tigecycline treating ventilator-associated pneumonia (VAP) due to S. maltophilia in comparison with fluoroquinolones. Methods This is a multicenter retrospective cohort study of patients admitted between January 2017 and December 2020 with the diagnosis of VAP caused by S. maltophilia receiving either tigecycline or fluoroquinolones as the definitive therapy ≥ 48 h. Clinical outcomes including 28-day mortality, clinical cure and microbiological cure were analyzed. Results Of 82 patients with S. maltophilia VAP included, 46 received tigecycline, and 36 received fluoroquinolones; 70.7% of patients had polymicrobial pneumonia, and the appropriate empiric therapy was applied to only 14.6% of patients. The overall 28-day mortality was 39%. Compared with patients receiving fluoroquinolones, tigecycline therapy resulted in worse clinical cure (32.6% vs. 63.9%, p = 0.009) and microbiological cure (28.6% vs. 59.1%, p = 0.045), while there was no statistical difference between 28-day mortality (47.8% vs. 27.8%, p = 0.105) in the two groups. Similar results were also shown in the inverse probability of treatment weighted univariable regression model and multivariable regression model. Conclusions The standard dose of tigecycline therapy was associated with a lower clinical and microbiological cure rate but not associated with an increased 28-day mortality in patients with S. maltophilia VAP compared with fluoroquinolones. Considering the unfavorable clinical outcomes, we therefore recommend against using the standard dose of tigecycline in treating S. maltophilia VAP unless new clinical evidence emerges. Supplementary Information The online version contains supplementary material available at 10.1007/s40121-021-00516-5.

Background Traditional therapies fail to cure most glioblastoma patients and the 5-year survival rate is less than 10%, highlighting need for new therapeutic approaches. The natural killer group 2 member D ligands (NKG2DLs) are highly expressed in glioblastomas and are considered promising targets for chimeric antigen receptor (CAR) T-cell therapy. The aim of this study was to investigate the effect of NKG2D-expressing CAR-T cells on glioblastomas and glioblastoma stem cells. Methods The expression of NKG2DLs was analyzed by flow cytometry and immunohistochemistry. NKG2D-BBz CAR, containing the extracellular domain of NKG2D, was constructed and delivered into T cells by lentiviral particles. In vitro cytotoxicity of the CAR-T cells was assessed by flow cytometry. Release of cytokine, perforin and granzyme B was quantified using enzyme-linked immunosorbent assay kits. The therapeutic efficacy of NKG2D-BBz CAR-T cells in vivo was evaluated using subcutaneous tumor models. The safety of the CAR was analyzed by investigating the effects on proliferation, apoptosis, and karyotype. Results Our data confirmed the high expression of NKG2DLs in human glioblastoma cells, cancer stem cells, and tumor samples. Further, the NKG2D-BBz CAR-T cells efficiently lysed glioblastoma cells and cancer stem cells in vitro and produced high levels of cytokines, perforin, and granzyme B. The CAR-T cells markedly eliminated xenograft tumors in vivo and did not exhibit significant treatment-related toxicity in the treated mice. The CAR expression also did not exert any obvious effects on cell proliferation, apoptosis, and genomic stability. Conclusion Our findings demonstrated that NKG2D CAR-T cells targeted glioblastoma cells and cancer stem cells in an NKG2D-dependent manner, supporting the use of CAR-T therapy in glioblastoma therapeutic strategies. Electronic supplementary material The online version of this article (10.1186/s40425-019-0642-9) contains supplementary material, which is available to authorized users.

Subarachnoid hemorrhage (SAH) is a cerebrovascular disease associated with high morbidity and mortality. CXCR4 provides neuroprotective effects, which can alleviate brain injury and inflammation induced by stroke. Previous studies have suggested that CXCR4 reduces the pyroptosis of LPS-stimulated BV2 cells. The purpose of this study was to evaluate the antipyroptosis effects and mechanisms of CXCR4 after SAH. SAH animal model was induced via endovascular perforation. A total of 136 male Sprague-Dawley rats were used. Recombinant human cysteine-X-cysteine chemokine ligand 12 (rh-CXCL-12) was administered intranasally at 1 h after SAH induction. To investigate the underlying mechanism, the inhibitor of CXCR4, AMD3100, was administered intraperitoneally at 1 h before SAH. The neurobehavior tests were assessed, followed by performing Western blot and immunofluorescence staining. The Western blot results suggested that the expressions of endogenous CXCL-12, CXCR4, and NLRP1 were increased and peaked at 24 h following SAH. Immunofluorescence staining showed that CXCR4 was expressed on neurons, microglia, and astrocytes. Rh-CXCL-12 treatment improved the neurological deficits and reduced the number of FJC-positive cells, IL-18-positive neurons, and cleaved caspase-1(CC-1)-positive neurons after SAH. Meanwhile, rh-CXCL-12 treatment increased the levels of CXCL-12 and CXCR4, and reduced the levels of NLRP1, IL-18, IL-1β, and CC-1. Moreover, the administration of AMD3100 abolished antipyroptosis effects of CXCL-12 and its regulation of CXCR4 post-SAH. The CXCR4/NLRP1 signaling pathway may be involved in CXCL-12-mediated neuronal pyroptosis after SAH. Early administration of CXCL-12 may be a preventive and therapeutic strategy against brain injury after SAH.

This is the first report of ceftazidime–avibactam resistance caused by the blaKPC-33 mutation through the D179Y variant during the treatment of blaKPC-2-positive Klebsiella pneumoniae-related infections in China. The blaKPC-33-containing K. pneumoniae was susceptible to meropenem–vaborbactam, cefepime–zidebactam, tigecycline, and polymyxin B. The blaKPC-33 gene was located on a 77 551-bp transformable plasmid harboring qnrS1 and blaLAP-2. Detecting blaKPC-33-positive K. pneumoniae clinical strains is important for infection control.

Background Hypoxic-ischemic encephalopathy (HIE) is a life-threatening cerebrovascular disease. Neuroinflammation plays an important role in the pathogenesis of HIE, in which microglia are key cellular mediators in the regulation of neuroinflammatory processes. Colony-stimulating factor 1 (CSF1), a specific endogenous ligand of CSF1 receptor (CSF1R), is crucial in microglial growth, differentiation, and proliferation. Recent studies showed that the activation of CSF1R with CSF1 exerted anti-inflammatory effects in a variety of nervous system diseases. This study aimed to investigate the anti-inflammatory effects of recombinant human CSF1 (rh-CSF1) and the underlying mechanisms in a rat model of HIE. Methods A total of 202 10-day old Sprague Dawley rat pups were used. HI was induced by the right common carotid artery ligation with subsequent exposure of 2.5-h hypoxia. At 1 h and 24 h after HI induction, exogenous rh-CSF1 was administered intranasally. To explore the underlying mechanism, CSF1R inhibitor, BLZ945, and phospholipase C-gamma 2 (PLCG2) inhibitor, U73122, were injected intraperitoneally at 1 h before HI induction, respectively. Brain infarct area, brain water content, neurobehavioral tests, western blot, and immunofluorescence staining were performed. Results The expressions of endogenous CSF1, CSF1R, PLCG2, protein kinase C epsilon type (PKCε), and cAMP response element-binding protein (CREB) were gradually increased after HIE. Rh-CSF1 significantly improved the neurological deficits at 48 h and 4 weeks after HI, which was accompanied by a reduction in the brain infarct area, brain edema, brain atrophy, and neuroinflammation. Moreover, activation of CSF1R by rh-CSF1 significantly increased the expressions of p-PLCG2, p-PKCε, and p-CREB, but inhibited the activation of neutrophil infiltration, and downregulated the expressions of IL-1β and TNF-α. Inhibition of CSF1R and PLCG2 abolished these neuroprotective effects of rh-CSF1 after HI. Conclusions Our findings demonstrated that the activation of CSF1R by rh-CSF1 attenuated neuroinflammation and improved neurological deficits after HI. The anti-inflammatory effects of rh-CSF1 partially acted through activating the CSF1R/PLCG2/PKCε/CREB signaling pathway after HI. These results suggest that rh-CSF1 may serve as a potential therapeutic approach to ameliorate injury in HIE patients.

Insects produce many peptide hormones that play important roles in regulating growth, development, immunity, homeostasis, stress, and other processes to maintain normal life. As part of the digestive system, the insect midgut is also affected by hormones secreted from the prothoracic gland, corpus allatum, and various neuronal cells; these hormones regulate the secretion and activity of insects' digestive enzymes and change their feeding behaviors. In addition, the insect midgut produces certain hormones when it recognizes various components or pathogenic bacteria in ingested foods; concurrently, the hormones regulate other tissues and organs. In addition, intestinal symbiotic bacteria can produce hormones that influence insect signaling pathways to promote host growth and development; this interaction is the result of long-term evolution. In this review, the types, functions, and mechanisms of hormones working on the insect midgut, as well as hormones produced therein, are reviewed for future reference in biological pest control.

The immunomodulatory drugs, lenalidomide and pomalidomide yield high response rates in multiple myeloma patients, but are associated with a high rate of thrombocytopenia and increased risk of secondary hematologic malignancies. Here, we demonstrate that the immunomodulatory drugs induce self-renewal of hematopoietic progenitors and upregulate megakaryocytic colonies by inhibiting apoptosis and increasing proliferation of early megakaryocytic progenitors via down-regulation of IKZF1. In this process, the immunomodulatory drugs degrade IKZF1 and subsequently down-regulate its binding partner, GATA1. This results in the decrease of GATA1 targets such as ZFPM1 and NFE2, leading to expansion of megakaryocytic progenitors with concomitant inhibition of maturation of megakaryocytes. The down-regulation of GATA1 further decreases CCND1 and increases CDKN2A expression. Overexpression of GATA1 abrogated the effects of the immunomodulatory drugs and restored maturation of megakaryocytic progenitors. Our data not only provide the mechanism for the immunomodulatory drugs induced thrombocytopenia but also help to explain the higher risk of secondary malignancies and long-term cytopenia induced by enhanced cell cycling and subsequent exhaustion of the stem cell pool.

Recently, mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) has emerged as an important key regulator of the stress-activated MAPK core signaling pathways. MAP4K2, also called Germinal Center Kinase (GCK), is predominantly and highly expressed in the germinal center of B cells. Recently we have shown that MAP4K2 knockdown in K- or N-RAS mutated MM cells induces MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins (Li et al. Blood 2021). Importantly, MAP4K2 silencing induces IKZF1 protein degradation without affecting IKZF1 mRNA level. Furthermore, IMiDs-resistant K-RAS Mut MM cells are sensitive to MAP4K2 inhibition induced IKZF1 degradation and cell growth suppression, suggesting that MAP4K2 inhibition overcomes IMiDs resistances in MM. To further validate MAP4K2 inhibition as a novel strategy to overcome IMiDs-resistance, we generated lenalidomide-resistant human myeloma cell lines. In this model, MM1S-LEN RES cells showed significantly decreased expression of CRBN protein compared to the parent cells. Accordingly, upon lenalidomide treatment, CRBN-mediated down-regulation of IKZF1, c-MYC, IKZF3 and IRF4 were abrogated in the MM1S-LEN RES cells. As expected, MM1S-LEN RES cells were resistant to lenalidomide induced growth inhibition in cell proliferation assay. In contrast, MAP4K2 inhibition using TL4-12 potently induced IKZF1, c-MYC, and IRF4 downregulation as well as cell proliferation inhibition, demonstrating that MAP4K2 regulates IKZF1 and cell growth independently of CRBN. These results indicate that MAP4K2 is a novel therapeutic target to overcome IMiDs-resistance MM. Iberdomide (CC-220) is an orally available IMiD® compound under development for the treatment of relapsed/refractory multiple myeloma. Previous biochemical and structural studies demonstrated that Iberdomide binds to cereblon with a higher affinity than lenalidomide or pomalidomide. Here, we evaluated the combination effects of Iberdomide with MAP4K2 silencing in MM. Tet-on sh-MAP4K2 lentivirus were introduced into RAS Mut MM cells to establish the inducible MAP4K2 knockdown cells upon doxycycline treatment. To address the combination effects, Tet-on sh-MAP4K2 RAS Mut MM cells were treated with doxycycline and different dosages of iberdomide. We found that MAP4K2 silencing strongly increased iberdomide-induced apoptosis (iberdomide alone vs. with MAP4K2 KD: 32% vs 92). Similar, in western blot assays, MAP4K2 silencing combined with Iberdomide significantly enhanced downregulation of IKZF1, c-MYC, and IRF4 compared to the iberdomide treatment alone. These data suggest that combination of iberdomide and MAP4K2 inhibition have synergetic anti-MM effects. Taken together, our findings demonstrate that MAP4K2 is a novel therapeutic target to bypass IMiDs resistance in RAS mutated MM. Combination of MAP4K2 inhibition with Iberdomide results in synergetic anti-cancer effects in MM, therefore could be a potent novel therapeutic regimen for patients with relapsed/refractory multiple myeloma. Disclosures Marcireau: Sanofi: Current Employment.

Glioblastoma multiform (GBM) is a highly aggressive brain tumor with poor life expectancy, and glioma stem cells (GSCs) are a small population of tumor cells existed in GBM, in which GSCs response to drive GBM recurrence, invasion and contribute to the anti-cancer resistance. GSCs have been identified and developed as a therapeutic target for GBM and can be used in drugs screening. Isocostunolide is a natural sesquiterpenoid and contained abundant resource in medicinal plants, but the anti-cancer efficacies of it against GSCs are still unexplored. In this investigation, the anti-tumor activity of isocostunolide against GSCs was investigated and the result demonstrated that it inhibited the growth of GSCs (GSC-3#, GSC-12#, GSC-18#) significantly with an IC50 value of 2.80μg/ml, 2.61μg/ml, 1.07μg/ml, respectively. In further mechanism study, isocostunolide inhibited GSCs cell proliferation, induced GSCs apoptosis significantly, as well as increased the proportion of the cleavage of caspase-3. The result suggested that isocostunolide induced GSCs apoptosis via the caspase dependent apoptotic pathway. Moreover, isocostunolide damaged GSCs colony formation capacity significantly and exhibited the anti-cancer efficacy against GSCs in vitro.

Transcription factors (TFs) are convergence points of signaling cascades that coordinate cell differentiation, proliferation, survival, and migration; and are commonly deregulated in solid and hematologic malignancies, including multiple myeloma (MM). Several recent studies indicate that the inhibition of TFs may lead to selective tumor cell death with little or no consequences for normal cells due to redundancy in signaling pathways. Nuclear hormone receptor (NHR)- TFs belong to the most common therapies in oncology today. In contrast, non-NHR-TFs have been considered 'un- druggable' until most recently.This review article summarizes advances of our knowledge on the complex composition of non-NHR-TFs and their binding to cognate DNA sequences that are propelling the development of novel strategies in MM.Protein-protein and protein-DNA- binding inhibitors, proteolysis- targeting chimeric molecules, and chromatin remodeling/epigenetic reader inhibitors are among the most promising novel compounds with a potentially high therapeutic index; they are likely to once more advance MM treatment strategies and improve patient outcome in the near future.

ClC-3, a member of the ClC chloride (Cl(-)) channel family, has recently been proposed as the primary Cl(-) channel involved in cell volume regulation. Changes in cell volume influence excitability, contraction, migration, pathogen-host interactions, cell proliferation, and cell death processes. In this study, expression and function of ClC-3 channels were investigated during epidermal stem cell (ESC) migration. We observed differential expression of CLC-3 regulates migration of ESCs. Further, whole-cell patch-clamp recordings and image analysis demonstrated ClC-3 expression affected volume-activated Cl(-) current (I Cl,Vol) within ESCs. Live cell imaging systems, designed to observe cellular responses to overexpression and suppression of ClC-3 in real time, indicated ClC-3 may regulate ESC migratory dynamics. We employed IMARIS software to analyze the velocity and distance of ESC migration in vitro to demonstrate the function of ClC-3 channel in ESCs. As our data suggest volume-activated Cl(-) channels play a vital role in migration of ESCs, which contribute to skin repair by migrating from neighboring unwounded epidermis infundibulum, hair follicle or sebaceous glands, ClC-3 may represent a new and valuable target for stem cell therapies.

Introduction: Next-generation sequencing revealed frequent mutations of the RAS/mitogen-activated protein kinase (MAPK) pathway, with mutations in NRAS, KRAS or BRAF in up to 50% of newly diagnosed MM patients1. The majority of the NRAS, KRAS and BRAF mutations occur in hotspots causing constitutive activation of the corresponding pathways2. Given the upstream activator role of Germinal Center Kinase (GCK) in the MAPK pathway, GCK might be an attractive therapeutic target in MM. Indeed, we recently discovered the critical role of GCK, also named mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), in MM cell survival and growth. Methods and Results: Our data show that GCK is higher expressed in RAS mutated MM cells compared to the wild type (WT) RAS MM cells. Silencing of GCK in RASmut MM cells (MM.1S and RPMI-8226) by using an inducible Tet-on-shGCK significantly decreased MM cell proliferation and induced cell death (Figure 1). In contrast, knockdown of GCK in RASwt MM cell lines (LP1 and U266) induced only modest inhibition of proliferation. The higher sensitivity to GCK knockdown in RASmut cells suggests that targeting GCK is especially effective in multiple myeloma which harbors RAS mutations. To exclude a potential off-target effect associated with GCK knockdown that led to the inhibition of MM proliferation, we set up an shGCK-resistant GCK allele (GCKshRNA-RES) by introducing mismatch mutations on the shRNA targeted sequences without changing the encoded amino acids. In the shGCK rescue experiment using GCKshRNA-RES we showed that all shRNA induced phenotypes (lack of growth, apoptosis and downstream effectors decrease) were corrected by the GCK resistant allele expression, ruling out the off-target hypothesis. Moreover, we expanded the in vivo studies of GCK knockdown on MM tumor progression. To monitor the tumor progression, we transduced MM.1S cells with firefly luciferase and established an inducible GCK knockdown system. Luciferase-expressing GCK inducible knockdown MM cells or non-targeting control shRNA (shCNTL) transduced MM cells were s.c. injected into SCID/Beige mice and the tumor progression was monitored by bioluminescence imaging. Doxycycline (for induction of shRNA) or vehicle treatment were started after the tumor was established on day 16 to induce shGCK and subsequently silence GCK expression. In contrast to the vehicle-treated MM.1S-Tet-on-shGCK or doxycycline-treated MM.1S-Tet-on-shCNTL tumors, doxycycline-treated animals bearing MM.1S-Tet-on-shGCK xenografts showed a significant inhibition (P Lysine 45 is critical for GCK kinase activity. Point mutation of K45A will completely abolish its kinase activity. We introduced K45A mutation into GCKshRNA-RES (GCKshRNA-RESK45A→ shGCK resistant and kinase dead GCK). Tet-on-shGCK with GCKshRNA-RES or GCKshRNA-RESK45A were co-transduced in MM.1S cells. As expected, the GCK knockdown effects were rescued by GCKshRNA-RES but not by the kinase-dead mutant GCKshRNA-RESK45A. In contrast to GCKshRNA-RES, GCKshRNA-RESK45A failed to stimulate MM cell proliferation, to suppress MM cells apoptosis and to restore the downstream effectors expression. Our findings demonstrated that GCK kinase activity is required for its function in myeloma cell physiology. Conclusion: Taken together, our findings provide a rationale for the clinical evaluation of targeting GCK in MM patients and the role of GCK in MM tumorigenesis as well as drug resistance. The subsequent development of small molecules inhibiting this pathway, such as GCK kinase inhibitors, will address the unmet need of developing targeted treatments for RASmut myeloma and potentially for other RASmut malignancies. References 1. Walker, B.A., et al. Mutational Spectrum, Copy Number Changes, and Outcome: Results of a Sequencing Study of Patients With Newly Diagnosed Myeloma. J Clin Oncol33, 3911-3920 (2015). 2. Xu, J., et al. Molecular signaling in multiple myeloma: association of RAS/RAF mutations and MEK/ERK pathway activation. Oncogenesis6, e337 (2017). Disclosures Marcireau: Sanofi: Employment. Lentzsch:Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Proclara: Consultancy; Abbvie: Consultancy; Clinical Care Options: Speakers Bureau; Sanofi: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Honoraria; International Myeloma Foundation: Honoraria; Karyopharm: Research Funding; Columbia University: Patents & Royalties: 11-1F4mAb as anti-amyloid strategy; Bayer: Consultancy.

Hamiltonella defensa is well known for its protective roles against parasitoids for its aphid hosts, but its functional roles in insect‐plant interactions are less understood. Thus, the impact of H. defensa infections on life‐history characters and the underlying genetic variation for the grain aphid, Sitobion avenae (Fabricius), was explored on three plants (i.e., wheat, oat, and rye). Compared to cured lines, H. defensa infected lines of S. avenae had lower fecundity on wheat and oat, but not on rye, suggesting an infection cost for the aphid on susceptible host plants. However, when tested on rye, the infected lines showed a shorter developmental time for the nymphal stage than corresponding cured lines, showing some benefit for S. avenae carrying the endosymbiont on resistant host plants. The infection of H. defensa altered genetic variation underlying its host S. avenea’s life‐history characters, which was shown by differences in heritabilities and genetic correlations of life‐history characters between S. avenae lines infected and cured of the endosymbiont. This was further substantiated by disparity in G‐matrices of their life‐history characters for the two types of aphid lines. The G‐matrices for life‐history characters of aphid lines infected with and cured of H. defensa were significantly different from each other on rye, but not on oat, suggesting strong plant‐dependent effects. The developmental durations of infected S. avenae lines showed a lower plasticity compared with those of corresponding cured lines, and this could mean higher adaptability for the infected lines.Overall, our results showed novel functional roles of a common secondary endosymbiont (i.e., H. defensa) in plant‐insect interactions, and its infections could have significant consequences for the evolutionary ecology of its host insect populations in nature.

Background Drought may become one of the greatest challenges for cereal production under future warming scenarios, and its impact on insect pest outbreaks is still controversial. To address this issue, life-history responses of the English grain aphid, Sitobion avenae (Fabricius), from three areas of different drought levels were compared under three water treatments. Results Significant differences were identified in developmental time, fecundity and adult weight among S. avenae clones from moist, semiarid and arid areas under all the three water treatments. Semiarid and arid area clones tended to have higher heritability for test life-history traits than moist area clones. We identified significant selection of water-deficit on the developmental time of 1st instar nymphs and adult weight for both semiarid and arid area clones. The impact of intermediate and severe water-stress on S. avenae’s fitness was neutral and negative (e.g., decreased fecundity and weight), respectively. Compared with arid-area clones, moist- and semiarid-area clones showed higher extents of adaptation to the water-deficit level of their respective source environment. Adult weight was identified as a good indicator for S. avenae’s adaptation potential under different water-stress conditions. After their exposure to intermediate water-deficit stress for only five generations, adult weight and fecundity tended to decrease for moist- and semiarid-area clones, but increase for arid-area clones. Conclusions It is evident from our study that S. avenae clones from moist, semiarid and arid areas have diverged under different water-deficit stress, and such divergence could have a genetic basis. The impact of drought on S. avenae’s fitness showed a water-level dependent pattern. Clones of S. avenae were more likely to become adapted to intermediate water-deficit stress than severe water-deficit stress. After continuous water-deficit stress of only five generations, the adaptation potential of S. avenae tended to decrease for moist and semiarid area clones, but increase for arid area clones. The rapid shift of aphids’ life-history traits and adaptation potential under drought could have significant implications for their evolutionary dynamics and outbreak risks in future climate change scenarios. Electronic supplementary material The online version of this article (10.1186/s12898-018-0173-0) contains supplementary material, which is available to authorized users.

Epidermal stem cells could contribute to skin repair through the migration of cells from the neighboring uninjured epidermis, infundibulum, hair follicle, or sebaceous gland. However, little is known about the factors responsible for the complex biological processes in wound healing. Herein, we will show that the attracting chemokine, SDF-1/CXCR4, is a major regulator involved in the migration of epidermal stem cells during wound repair. We found that the SDF-1 levels were markedly increased at the wound margins following injury and CXCR4 expressed in epidermal stem cells and proliferating epithelial cells. Blocking the SDF-1/CXCR4 axis resulted in a significant reduction in epidermal stem cell migration toward SDF-1 in vitro and delayed wound healing in vivo, while an SDF-1 treatment enhanced epidermal stem cell migration and proliferation and accelerated wound healing. These results provide direct evidence that SDF-1 promotes epidermal stem cell migration, accelerates skin regeneration, and makes the development of new regenerative therapeutic strategies for wound healing possible.

Background Hypertrophic scarring (HS) is a severe disease, and results from unusual wound healing. Col1A1 could promote the hypertrophic scar formation, and the expression of Col1A1 in HS tissue was markedly higher than that in the normal. In present study, we aimed to identify miRNAs as post-transcriptional regulators of Col1A1 in HS. Methods MicroRNA-98 was selected as the key miRNA comprised in HS. The mRNA levels of miR-98 in HS tissues and the matched normal skin tissues were determined by qRT-PCR. MTT and flow cytometry were used to determine the influence of miR-98 on cell proliferation and apoptosis of HSFBs, respectively. Col1A1 was found to be the target gene of miR-98 using luciferase reporter assay. Luciferase assay was performed to determine the relative luciferase activity in mimic NC, miR-98 mimic, inhibitor NC and miR-98 inhibitor with Col1A13′-UTR wt or Col1A13′-UTR mt reporter plasmids. The protein expression of Col1A1 in HSFBs after transfection with mimic NC, miR-98 mimic, inhibitor NC and miR-98 inhibitor were determined by western blotting. Results The mRNA level of miR-98 in HS tissues was much lower than that in the control. Transfection of HSFBs with a miR-98 mimic reduced the cell viability of HSFBs and increased the apoptosis portion of HSFBs, while inhibition of miR-98 increased cell viability and decreased apoptosis portion of HSFBs. miR-98 inhibitor increased the relative luciferase activity significantly when cotransfected with the Col1A1-UTR reporter plasmid, while the mutant reporter plasmid abolished the miR-98 inhibitor-mediated increase in luciferase activity. Western blotting revealed that overex-pression of miR-98 decreased the expression of Col1A1. Conclusions Overexpression of miR-98 repressed the proliferation of HSFBs by targeting Col1A1.

Phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating the mechanistic target of rapamycin (mTOR). Because mTOR signaling is required for initiation of messenger RNA translation, we hypothesized that cotargeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. We found that a novel PI3K δ isoform inhibitor TGR-1202, but not the approved PI3Kδ inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells. TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc-dependent transcription. The synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kδ inhibitors, inhibited casein kinase-1 ε (CK1ε). Targeting CK1ε using a selective chemical inhibitor or short hairpin RNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. These results suggest that TGR-1202 is a dual PI3Kδ/CK1ε inhibitor, which may in part explain the clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1ε should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc.

Objective Epidermal stem cells (ESCs) play a critical role in wound healing, but the mechanism underlying ESC proliferation is not well defined. Here, we explore the effects of RhoA on ESC proliferation and the possible underlying mechanism. Methods Human ESCs were enriched by rapid adhesion to collagen IV. RhoA(+/+)(G14V), RhoA(-/-)(T19N) and pGFP control plasmids were transfected into human ESCs. The effect of RhoA on cell proliferation was detected by cell proliferation and DNA synthesis assays. Induction of PKN1 activity by RhoA was determined by immunoblot analysis, and the effects of PKN1 on RhoA in terms of inducing cell proliferation and cyclin D1 expression were detected using specific siRNA targeting PKN1. The effects of U-46619 (a RhoA agonist) and C3 transferase (a RhoA antagonist) on ESC proliferation were observed in vivo. Results RhoA had a positive effect on ESC proliferation, and PKN1 activity was up-regulated by the active RhoA mutant (G14V) and suppressed by RhoA T19N. Moreover, the ability of RhoA to promote ESC proliferation and DNA synthesis was interrupted by PKN1 siRNA. Additionally, cyclin D1 protein and mRNA expression levels were up-regulated by RhoA G14V, and these effects were inhibited by siRNA-mediated knock-down of PKN1. RhoA also promoted ESC proliferation via PKN in vivo. Conclusion This study shows that the effect of RhoA on ESC proliferation is mediated by activation of the PKN1-cyclin D1 pathway in vitro, suggesting that RhoA may serve as a new therapeutic target for wound healing.

In abnormal skin wound healing, hypertrophic scars (HS) are characterized by excessive fibroblast hypercellularity and an overproduction of collagen, leading to atypical extracellular matrix (ECM) remodeling. Although the exact mechanisms of HS remain unclear, decreased HS fibroblast (HSFB) apoptosis and increased proliferation are evident in the development of HS. In this study, the contribution of the second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein (IAP)-binding protein with a low isoelectric point (pI) (Smac/DIABLO), an apoptosis-promoting protein released from the mitochondria, was investigated in human normal skin and HSFB cultures. The expression of Smac/DIABLO is usually decreased in many malignant tumors compared with normal tissues. Immunohistochemical analysis of skin tissues and the western blot analyses of fibroblasts revealed that the expression of Smac/DIABLO was lower in HS tissues compared with normal skin tissues. Of note, adenovirus-mediated Smac/DIABLO overexpression in the cultured HSFBs significantly reduced cell proliferation, as detected by the cell counting kit-8, and increased caspase-3 and -9 activity, as detected by spectrofluorimetry. In addition, it increased apoptosis, as detected by fluorescence-activated cell sorting (FACS). Furthermore, we found that the silencing of Smac with siRNA in the HSFBs induced a noticeable decrease in caspase-3 and -9 activity, leading to a significant reduction in apoptosis. In addition, the mRNA expression of type I and III pro-collagen detected in the HSFBs was significantly increased following the silencing of Smac with siRNA and was inhibited following Smac/DIABLO overexpression, as shown by real-time RT-PCR. In conclusion, Smac/DIABLO decreases the proliferation and increases the apoptosis of HSFBs. To our knowledge, the data from our study suggest for the first time that Smac/DIABLO is a novel therapeutic target for HS.

This study assessed the roles of Tau gene short tandem repeats (STRs) nested within intron 1 (rs5820604 and rs10675722) and Apolipoprotein E (ApoE) gene polymorphisms in 70 late-onset Alzheimer's disease (AD) subjects, 70 vascular dementia (VD) subjects, and 70 normal controls (NCs) in Guizhou Han population. Our data suggest that the Tau gene STR loci rs5820604 (CA)(19) allele may increase the risk of AD, whereas the (CA)(16) allele may be protective. The Tau gene STR loci rs10675722 (TA)(17) allele may increase the risk for AD and VD. In addition, a synergistic effect of polymorphisms in ApoE ε4 and Tau gene STR loci rs5820604 (CA)(19) allele was found in the pathogenesis of AD (p = .025, OR [odds ratio] = 3.178, 95% CI [confidence interval] = 1.156-8.741).

eIF4E is the key regulator of protein translation and critical for translation. The oncogenic potential of tumorigenesis, which is highly contingent on cap-dependent eIF4E, also arises from the critical role in the nuclear export and cytosolic translation of oncogenic transcripts. Inhibition of Exportin1 (XPO1), which is the major nuclear export protein for eIF4E-bound oncoprotein mRNAs, results in decreased tumor cell growth in vitro and in vivo, suggesting that eIF4E is critical in multiple myeloma. Indeed, we found that eIF4E is overexpressed in myeloma cell lines and primary myeloma cells compared with normal plasma cells. Although stable overexpression of eIF4E in multiple myeloma cells significantly increases tumorigenesis, knockdown of eIF4E impairs multiple myeloma tumor progression in a human xenograft mouse model. Using a tet-on–inducible eIF4E-knockdown system, eIF4E downregulation blocks multiple myeloma tumor growth in vivo, correlating with decreased eIF4E expression. Further overexpression and knockdown of eIF4E revealed that eIF4E regulates translation of mRNAs with highly complex 5′-untranslated regions, such as c-MYC and C/EBPβ, and subsequently proliferation in multiple myeloma cells, but not in nonmalignant bone marrow stromal cells. Because many transcription factors that are critical for multiple myeloma proliferation exhibit a higher dependency on protein translation, eIF4E is an ideal and selective tool to target multiple myeloma cell growth. Mol Cancer Ther; 15(4); 711–9. ©2016 AACR.

Hypertrophic scar (HS) remains a major problem in plastic surgery. In order to explore the regulative effect of phosphatase and tensin homolog (PTEN) on HS, PTEN and AKT expression was detected by reverse transcription PCR, immunohistochemistry and western blot. Adenovirus-mediated PTEN overexpression in cultured hypertrophic scar fibroblasts (HSFBs) and normal skin fibroblasts was also introduced to evaluate its biological function. Our results showed that PTEN expression was significantly decreased in HS whereas p-Akt level was significantly higher in HS compared with normal skin (P < 0.01). Furthermore, we found that adenovirus-mediated PTEN overexpression led to decreased AKT activation, and significantly reduced cell proliferation and collagen synthesis of HSFBs, while increased the apoptosis. Taken together, these data suggest that PTEN inhibits proliferation and function of HSFBs through AKT pathway. Our results reveal a novel biological role for PTEN/AKT pathway in HS and suggest PTEN as a potential therapeutic target for HS treatment.

Mesenchymal stem cells (MSCs) are promising candidates for skin wound repair due to their capabilities of accumulating at wounds and differentiating into multiple types of skin cells. However, the underlying mechanisms responsible for these processes remain unclear. In this study, we found that osteopontin (OPN) stimulated the migration of MSCs in vitro, and observed the recruitment of endogenous MSCs to a skin wound and their differentiation into keratinocytes and endothelial cells. In OPN knock-out mice, the recruitment of MSCs to the skin wound was significantly inhibited, and wound closure was hampered after an intradermal injection of exogenous MSCs compared to wild-type mice. Consistent with these observations, the expressions of adhesion molecule CD44 and its receptor E-selectin were significantly decreased in the lesions of OPN knock-out mice compared with wild-type mice suggesting that OPN may regulate the migration of MSCs through its interactions with CD44 during skin wound recovery. In summary, our data demonstrated that OPN played a critical role in activating the migration of MSCs to injured sites and their differentiation into specific skin cell types during skin wound healing.

Introduction: c-Myc is a master transcription factor and one of the most frequently altered genes across a vast array of human cancers including diffuse large B-cell lymphoma (DLBCL), and is thus an attractive therapeutic target . However, no direct inhibitor of c-Myc has been successfully developed for the treatment of any cancer. The c-Myc protein has a short half-life of less than 30 minutes , and the complex secondary structures in the 5' untranslated region (UTR) of MYC mRNA make its translation highly dependent on the eukaryotic translation initiation factor 4F (eIF4F) . eIF4F exists as a complex comprised of the eIF4E, eIF4A, and eIF4G subunits. eIF4E can be sequestered by 4E-BP1, which acts as a "brake" for initiation of mRNA translation . Hyper-phosphorylation of 4E-BP1, caused by upstream signals such as mTORC1, leads to release of eIF4E from 4E-BP1, assembly of the eIF4F complex, and robust mRNA translation. Surprisingly, neither FDA approved mTORC1 inhibitors nor the investigational mTORC1/mTORC2 inhibitor MLN0128 has demonstrated adequate activity in aggressive lymphoma. The therapeutic effects of mTOR inhibition in c-Myc driven aggressive lymphoma remain poorly understood. Recognizing phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating mTOR, we hypothesized that co-targeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. Since both PI3K and proteasome are proven drug targets in blood cancer, such co-targeting strategy may be expeditiously studied in clinical trials for c-Myc driven aggressive lymphoma. Methods: Cytotoxicity was studied in lymphoma cell lines and primary lymphoma cells using Cell TiterGlo (Promega®). The Bliss additivism model was used to determine the expected inhibition of cell growth and the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Expression of c-Myc was investigated at the translation and transcription levels, using a combination of Western blot, qPCR, and a bi-cistronic luciferase reporter we developed to study cap dependent translation. Gene expression profiling (GEP) studies were conducted using RNAseq, and analyzed by the Fisher t-test and running enrichment score (RES) between different treatment groups. Mechanisms of synergy were determined through interrogating the effects of small molecule inhibitors and shRNA targeting regulators of various regulators of 4E-BP1. Structural studies of TGR-1202 were performed by in silico docking, and validated by synthesis of novel analogs of TGR-1202. Activity of TGR-1202 on CK1 epsilon was studied by kinome profiling (Reaction Biology®), cell free kinase assay of CK1e (Promega®), and cell based assay of CK1e autophosphorylation. Results: We found that a novel PI3K delta isoform inhibitor TGR-1202, but not the approved PI3Kd inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells (Figure 1). TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of eIF4E-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc dependent transcription (Figure 2). Furthermore, the synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kd inhibitors, was active against casein kinase-1 (CK1) epsilon (Figure 3). Targeting CK1e using a selective chemical inhibitor or shRNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. Conclusion: These results suggest that TGR-1202 is a first-in-class dual PI3Kd/CK1e inhibitor, which may in part explain the preliminary clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1e should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc. Disclosures Lentzsch: BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria. O'Connor:Seattle Genetics: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding; Spectrum: Research Funding; Spectrum: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; TG Therapeutics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Celgene: Research Funding.

IFNγ signaling plays a critical role in the pathogenesis of GVHD. In this study, we observed that LPS-maturated bone marrow-derived dendritic cells (BMDCs) lacking IFNγ receptor (IFNγR, GRKO) or signal transducer and activator of transcription 1 (STAT1KO) had increased expression of major histocompatibility complex class II (MHC II), CD86, CD80, and enhanced allo-stimulatory capacity. This was further confirmed using fully MHC-mismatched bone marrow transplantation (BMT) studies. APC of GRKO or STAT1KO recipients had increased MHC II expression, which was associated with enhanced activation and expansion of donor CD4 and CD8 T cells and subsequently accelerated GVHD mortality compared to wild type (WT) controls. This increased GVHD mortality and increased MHC II expression on host APCs was further observed in the absence of recipient conditioning in the B6→CB6F1 mouse model. This was associated with increased presentation of host derived endogenous Eα52-68 peptide via I-Ab on recipient CD11c+ cells as detected by staining with the YA-e antibody. Furthermore, we could demonstrate that absence of IFNγR in BMDC promotes presentation Eα52-68 peptide and subsequently elicits pronounced activation, expansion and Th1 differentiation of TEa-TCR-tg CD4 T cells which recognize the Eα52-68 peptide presented by I-Ab. Next, we assessed the impact of this pathway on presentation of exogenous antigens. Interestingly, when lysate prepared from BALB/c splenocytes was incubated with BMDCs from B6 mice, Y-Ae expression on STAT1-/- BMDCs was significantly reduced compared to wild type BMDCs allowing us to hypothesize that IFNγ/STAT1 signaling may play an important role in promoting presentation of exogenous antigen while suppressing presentation of endogenous antigen. To further confirm this hypothesis, we used ovalbumin (OVA) as a second model antigen. To assess the impact of IFNγ/STAT1 signaling on presentation of exogenous antigen, WT, GRKO or STAT1KO BMDC were directly pulsed with OVA. To address the role in endogenous antigen presentation we studied act-mOVA-transgenic wildtype, act-mOVA.GRKO or act-mOVA.STAT1KO BMDCs. Transgenic OT-II CD4 T cells express a TCR specific for the OVA peptide 323-33 presented by I-Ab. The proliferation/activation of OT II T cells was monitored by flow cytometer as readout for effective Ag presentation. Our data demonstrated that IFNγR- or STAT1-deficient BMDCs loaded with exogenous intact OVA protein were compromised in promoting OT II proliferation. In contrast, responder OT-II CD4 T cells proliferated much more vigorously when stimulated with IFNγR/STAT1-deficient m-Act-OVA BMDCs compared to controls. We further observed significantly impaired OT-II cell proliferation in IFNγR or STAT1-deficient mice immunized with OVA indicating impaired presentation of exogenous antigens. However, OT-II CD4 T cells injected into lethally irradiated act-mOVA.STAT1KO transgenic mice proliferated more robustly and displayed increased Th1 differentiation compared to control mice when tested 3 days after OT II administration. We next started to assess several key factors (Ii [invariant chain, CD74], Cathepsin S [CTSS], H2-M, CIITA and MARCH1), known to be involved in the process of MHC class II antigen presentation and MHC II expression. We found retention of Invariant chain (CD74) expression as well as reduced CTSS and H2M expression in GRKO or STAT1KO BMDC following LPS-maturation. Furthermore, we observed significantly reduced lysosome formation/function in STAT1KO BMDCs compared to wild type BMDCs after LPS maturation. These data suggest that exogenous protein-derived peptide exchange in the MHCII compartment (MIIC) is impaired in STAT1KO BMDCs. Immature and LPS-maturated STAT1-/-BMDCs had significantly increased autophagy, which could explain enhanced endogenous Ag presentation since autophagy has been demonstrated to be critical in MHC II Ag presentation of cytoplasmic constituents. Finally, we found evidence of enhanced MHC II synthesis as supported by increased CIITA mRNA expression and conversely reduced MHC II degradation as indicated by reduced MARCH1 expression. In summary our data suggest that absence of IFNγR/STAT1 signaling in DC leads to abnormal surface MHC II turnover, promotes presentation of endogenous peptides and concomitantly impairs processing and presentation of exogenous antigens. Disclosures Lentzsch: BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria.

Introduction: Bay 61-3606 is a cell-permeable imidazopyrimidine compound that acts as a potent, ATP-competitive, reversible, and highly selective inhibitor of Syk tyrosine kinase activity with no inhibitory effect against Btk, Fyn, Itk, Lyn, and Src. BAY 61-3606 has been also shown to inhibit Syk-mediated cellular functions such as glucose-tyrosine phosphorylation of I κ B α and p65 nuclear translocation. It further exhibits a good oral bioavailability and in vivo efficacy in rat models. Recently, Bay 61-3606 was found to inhibit cell proliferation and SDF-1a-induced migration of MM cells (1). Based on these promising preliminary data we further investigated the potential of Bay 61-3606 as new anti-MM agent. Methods and Results: Bay 61-3606, at concentrations as low as 10 nM, induced significant (p Conclusion: In summary, our results demonstrate that Bay 61-3606 has anti-MM effects by inducing cell cycle arrest and apoptosis, and this effect is independent of SYK inhibition. Similar to IMiD® compounds, Bay 61-3606 induces IKZF1 and IKZF3 protein degradation in MM cells. More importantly, Bay 61-3606 has effects on both IMiD®-sensitive and resistant myeloma cells and may represent a novel potent anti-MM agent. References: (1). Koerber RM, et al. Exp Hematol Oncol. 2015. Disclosures Lentzsch: BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria.

(-)-Epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epicatechin (EC) were found to have different cytotoxicities to human colon carcinoma LoVo cells. EGCG and EGC suppressed the growth of LoVo cells in dose-dependent manner; ECG and EC, however, had no obvious effects. At lower concentrations, EC did seem to promote slight proliferation. Similarly, EGCG and EGC, rather than ECG and EC, were found to induce apoptosis in LoVo cells. Moreover, EGCG, EGC, and ECG arrested G(1) phase in the cell cycle progression, whereas EC resulted in S phase arrest. We suggest that there are differences in the cytotoxicities of the four catechins to LoVo cells. They exert the effects by inducing apoptosis and regulating the cell cycle.

It has been shown that overexpression and/or activation of eukaryotic initiation factor 4E (eIF4E) induces a malignant phenotype by regulating oncogenic protein synthesis. Aberrant control of protein synthesis contributes to lymphoma genesis, opening up possibilities of innovative therapeutics by targeting the translational machinery. In multiple myeloma (MM), understanding the mechanisms that control protein synthesis is emerging as an exciting new research area with significant potential for developing innovative therapies. Recently, eIF4E has been discovered as a critical regulator in protein synthesis. This review will summarize the role and regulatory mechanism of eIF4E in multiple myeloma, discuss the potential usage of mRNA translation controlling drugs for multiple myeloma treatment, and provide new leads for novel strategies of MM therapy.

Hypertrophic scars (HS) are skin disorders which occur after wounding and thermal injury. Our previous studies have suggested that secreted frizzled-related protein 2 (SFRP2) is involved in HS formation and that the suppression of SFRP2 promotes apoptosis of hypertrophic scar fibroblasts (HSFBs). However, the mechanisms have not been clarified. Previous studies revealed that Slug expression inhibits cell apoptosis, in vitro and in vivo, and SFRP2 regulates the expression of Slug in cervical cancer cells. In the present study, we quantified differential expression levels of expression of SFRP2 and Slug in HS and normal skin tissues by immunohistochemistry, both of which have important anti-apoptosis roles. Furthermore, a short hairpin RNA approach was adopted to investigate the potential function of SFRP2 and Slug in HSFB apoptosis. Cell apoptosis was detected using fluorescence-activated cell sorting and Caspase-3 activity was assayed by spectrophotometry. This study demonstrates that SFRP2 expression, as well as Slug, is dramatically up-regulated in HS relative to normal skin tissues, and the Slug expression is positively correlated with SFRP2. Slug expression was down-regulated in SFRP2-deficient cells, and the down-regulation of Slug expression increased sensitivity to apoptosis which was induced through a caspase-3-dependent pathway. The infected cells with reduced levels of Slug were tested for the expression of apoptosis-related genes (Bcl-2, Bax and PUMA) which were previously identified as Slug targets. Bcl-2 expression was down-regulated in Slug-deficient cells. In conclusion, SFRP2 appears to interact with Slug to affect the apoptosis of hypertrophic scar fibroblasts.

Background : We have recently shown that Matrix metalloproteinase-13 (MMP-13) is highly expressed in human multiple myeloma (MM) cells and MMP-13 increases osteoclast (OCL) fusion and bone resorption independently of its metalloproteinase activity. In a mouse model of MM bone disease, knockdown (KD) of MMP-13 in MM cells inhibited development of bone lytic lesions, suggesting that MMP-13 is a potential therapeutic target for the treatment of MM bone disease (MMBD). However, the effects of MMP-13 on the MM bone marrow (BM) niche and underlying mechanism by which MMP-13 induces OCL fusion remains undefined. Methods and Results: To delineate the effects of MMP-13 on MM BM microenvironment, we investigated the effects of MMP-13 on cell proliferation and differentiation of pre-OCL, pre-osteoblast and MM cells. WT or Mmp-13-/- mouse CD11b+ BM cells were cultured with or without MMP-13 for 3 days and pre-OCL proliferation was evaluated by WST-1 assay. Neither MMP-13 treatment nor Mmp-13 deficiency affected pre-OCL proliferation, indicating MMP-13 induces OCL activation rather by promoting cell fusion than by affecting cell proliferation. To test the effects of MMP-13 on osteoblast (OB) differentiation and mineralization, mouse pre-OB cell line MC3T3-E1 and primary pre-OB cells from WT or Mmp-13-/- mice BM were cultured with or without MMP-13 for 7-14d. Neither exogenous MMP-13 nor Mmp-13 knockout affected OB differentiation or mineralization in vitro. Consistent with the in vitro observation, our Rag2-/- mouse 5TGM1 MMBD model provided in vivo evidence that knockdown of MMP-13 in MM cells does not affect MM induced OB inhibition or impairment of bone formation. Further, the effects of MMP-13 on MM cell proliferation were also assessed both in vitro and in vivo. Treatment of human MM cell lines RPMI-8266, OPM2 and MM.1S, as well as mouse MM cell line 5TGM1 with MMP-13 for up to 3 days did not induce in vitro cell proliferation. Similarly, MM cell sensitivity against anti-MM agents bortezomib and pomalidomide was not affected by MMP-13 in vitro. But, MMP-13 KD in mouse 5TGM1 MM cell line caused significantly less in vivo tumor burden in Rag2-/- intratibial MMBD model compared to 5TGM1 empty vector control cells, which we speculate occurs as a consequence of effects secondary to decreased OCL activation within the bone microenvironment following MMP-13 silencing. Our previous experiments showed that MMP-13 induces OCL fusion independent of its enzymatic activity, however the underlying mechanism is unknown. To further analyze the mechanism of action immunofluorescence staining and cell fraction-immunoblotting assay demonstrated that extracellular MMP-13 undergoes cell surface binding and subsequent active intracellular translocation into the perinuclear area, further supporting the enzymatic activity independent mechanism of MMP-13 on OCL induction. Furthermore, analysis of MMP-13-induced signaling pathways mediating osteoclastogenesis revealed that MMP-13 activates multiple cell signaling pathways including MAPK, PI3K and NF-kappaB independent of its enzymatic activity. Interestingly, the noncanonical NF-kappaB pathway is selectively activated by either MMP-13 WT or an E223A catalytically-inactive mutant, but not any by any other MMPs or MMP-13 truncated non-functional mutants, indicating that signaling through the noncanonical NF-kappaB pathway in OCL is specific for MMP-13 and may play a role in MMP-13-driven OCL fusion. Further studies are underway to screen for MMP-13 receptors and binding partners in an effort to delineate the processes underlying MMP-13 signaling. Conclusion: Our results demonstrate that MM produced MMP-13 induces OCL activation and bone resorption without affecting OB inhibition and bone formation impairment as well as anti-neoplastic response of MM cells. MMP-13 silencing in MM protects bone structure, and inhibits MM progression in vivo, which may be secondary to decreased OCL activation. MMP-13 specifically acts on OCL fusion. Signaling study showed noncanonical NF-kappaB signaling is selectively activated by MMP-13 independently of its enzymatic activity, and may be critical for MMP-13-induced OCL fusion and activation. Further screening the potential cellular receptor and intracellular transportation mechanism of MMP-13 in OCL is ongoing. Disclosures Roodman: Amgen: Consultancy; Eli Lilly: Research Funding. Lentzsch:Axiom: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Introduction: Previously we have shown that the immune modulatory drugs (IMiDs) downregulate GATA1 and PU.1 resulting in maturational arrest of granulocytes with accumulation of immature myeloid precursors and subsequent neutropenia. Our studies further revealed that similar to MM cells cereblon (CRBN) is critical for the mediation of the effects of IMiDS in hematopoietic stem cells (HSCs) and associated with decrease of IKZF1-dependent transcription factors such as GATA1 and PU.1, which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Here we investigated the mechanism how IMIDs induce degradation of IKZF1 and confirmed our studies in vivo by using the humanized NOD/SCID/Gamma-c KO (NSG) mouse model. Methods and Results After we had shown that knockdown of CRBN in HCS mediates resistance to IMIDs (2014 ASH abstract 418) we assessed the impact of IKZF1 inhibition using two different approaches. First, we knocked down IKZF1 expression in CD34+ cells by shRNA lentivirus transduction. As expected, IKZF1 knockdown in CD34+ cells mimicked the effects of IMiDs resulting in increased CD34+ cell proliferation, CD33+ cell expansion (flow cytometry) and shift of lineage commitment from BFU-E to CFU-G (colony assay). Knockdown of IKZF1 was associated with decreased GATA1 and PU.1 expression at both mRNA and protein levels. Next, we generated a mutant IKZF1 by substituting Glutamine Q146 to Histidine, which abrogates IKZF1 ubiquitination induced by CRBN. CD34+ cells were transduced with lentiviral constructs to overexpress IKZF1-WT or IKZF1-Q146H. POM failed to induce IKZF1 degradation in IKZF1-Q146H-OE CD34+ cells, indicating CRBN binding to IKZF1 and subsequent ubiquitination is critical in this process. Functional assays further confirmed that IKZF1-Q146H CD34+ cells were resistant to POM induced CD33+ cell expansion and shift in lineage commitment from BFU-E to CFU-G. Since conventional mouse models are not applicable to test IMIDs in vivo due to the fact that IMIDs do not bind to mouse CRBN (Kronke, Fink et al. 2015), we established a humanized mouse model resembling human hematopoiesis. In this model, NOD/SCID/Gamma-c KO (NSG) mice received human fetal thymus grafts and 105 CD34+ fetal liver cells to generate human hematopoiesis including functional T-cells. After establishing human hematopoiesis mice were injected with POM (0.3 mg/kg) i.v every 2 days for 3 weeks. Analysis of bone marrow revealed that POM treatment significantly induced granulocyte/macrophage progenitor cells (CD34+ CD38+ CD45RA+ cells) at the expense of common lymphoid progenitors (CD34+ CD10+ cells). The shift into myelopoiesis is consistent with our in vitro finding that IMiDs affect lineage commitment. Conclusion: In summary, our results demonstrate that IMiDs affect CD34+ cell fate via CRBN and IKZF1 mediated mechanism. These results will be helpful to elucidate the mechanism of IMiDs on lineage commitment and maturation in HSCs. Also establishment of the humanized xenograft mice model may provide an advanced platform for the analysis of human hematopoiesis and human immune responses to IMiDs as well development of secondary hematologic malignancies in vivo. Disclosures Lentzsch: Axiom: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

We identified nocodazole as a potent antimyeloma drug from a drug screening library provided by the Multiple Myeloma Research Foundation. Nocodazole is a benzimidazole that was originally categorized as a broad-spectrum anthelmintic drug with antineoplastic properties. We found that nocodazole inhibited growth and induced apoptosis of primary and multiresistant multiple myeloma cells cultured alone and in the presence of bone marrow stromal cells. Nocodazole caused cell-cycle prophase and prometaphase arrest accompanied by microtubular network disarray. Signaling studies indicated that increased expression of Bim protein and reduced X-linked inhibitor of apoptosis protein and Mcl-1L levels were involved in nocodazole-induced apoptosis. Further investigation showed Bcl-2 phosphorylation as a critical mediator of cell death, triggered by the activation of c-jun-NH2 kinase (JNK) instead of p38 kinase or extracellular signal–regulated kinases. Treatment with JNK inhibitor decreased Bcl-2 phosphorylation and subsequently reduced nocodazole-induced cell death. Nocodazole combined with dexamethasone significantly inhibited myeloma tumor growth and prolonged survival in a human xenograft mouse model. Our studies show that nocodazole has potent antimyeloma activity and that targeting the microtubular network might be a promising new treatment approach for multiple myeloma. Mol Cancer Ther; 10(10); 1886–96. ©2011 AACR.

Although the mechanisms by which the HTLV-I Tax oncoprotein activates the two NF-κB signaling pathways have been well studied, there is still no convincing evidence demonstrating a functional role for NF-κB in the Tax-mediated tumorigenesis or HTLV-I-mediated adult T-cell leukemia/lymphoma (ATL). Moreover, how Tax is negatively regulated by cellular factors has not yet been explored. Using various transgenic mice and cells deficient in different key IKK/NF-κB signaling components, we have defined the differential but cooperative roles of the canonical and noncanonical NF-κB pathways in Tax-mediated tumorigenesis. One function of non-canonical NF-κB activation is to repress expression of the wwox tumor suppressor gene, which in turn facilitates Tax-induced canonical NF-κB activation. Mechanistic studies indicate that WWOX blocks Tax-induced IKKalpha recruitment and subsequent RelA phosphorylation. Furthermore, we have identified PDLIM2, an essential terminator of canonical NF-κB activation, as a negative regulator of Tax. PDLIM2 directly binds to and shuttles Tax from its activation sites to the nuclear matrix for ubiquitination-mediated degradation, thereby suppressing the tumorigenicities of HTLV-I- or Tax-transduced cells. Interestingly, PDLIM2 expression is epigenetically downregulated in HTLV-I-transformed T cells and primary ATL cells. These studies suggest that the counterbalance between PDLIM2 and HTLV-I may determine the ATL leukemogenesis and provide the first line of genetic evidence demonstrating the functional significance of NF-κB in Tax-mediated tumorigenesis. These studies also define, for the first time, a delicate cooperation of the two pro-oncogenic NF-κB pathways in tumorigenesis and a functional role of the WWOX tumor suppressor in NF-κB regulation and viral tumorigenesis.

Human T-cell leukemia virus type I (HTLV-I) encodes a Tax oncoprotein that has crucial roles in both virus replication and cell transformation. Our recent studies suggest that the counterbalance between HTLV-I/Tax and PDZ-LIM domain-containing protein PDLIM2 may determine the outcome of HTLV-I infection. Although HTLV-I represses PDLIM2 epigenetically and specifically in transformed cells, PDLIM2 shuttles Tax into the nuclear matrix for ubiquitination-mediated proteasomal degradation, thereby suppressing the transforming ability of HTLV-I. Here, we have further shown that PDLIM2 binds to Tax directly, which was mediated by a putative α-helix motif of PDLIM2 at amino acids 236–254. Consistently, selective disruption of this short-helix crippled PDLIM2 in shutting Tax to the nuclear matrix for ubiquitination-mediated degradation, therefore, PDLIM2 lost the ability in tumor suppression. Although the C-terminal LIM domain of PDLIM2 was not required for Tax binding, it was important for PDLIM2 to interact with the nuclear matrix. Accordingly, the LIM domain was essential for PDLIM2-mediated Tax repression. On the contrary, the N-terminal PDZ domain of PDLIM2 was dispensable for all these events, although the PDZ domain was involved in PDLIM2 binding to cytoskeleton. These studies dissect functional sequences within PDLIM2 and their distinct roles in Tax regulation.

Hypertrophic scars result from a dysregulated process in wound healing. Although the basic mechanism is unclear, increased proliferation and decreased cell apoptosis are noticed in the development of hypertrophic scar. In previous study, we found that secreted frizzled-related protein 2 (SFRP2), which was associated with cell proliferation, apoptosis, and differentiation, was dramatically upregulated in hypertrophic scar (HS) tissue. In this study short hairpin RNA (shRNA) targeting SFRP2 was employed to characterize SFRP2 function in hypertrophic scar-derived fibroblasts (HSFb). Cell proliferation was assessed by MTT, dynamic growth curves, and BRDU assays. Meanwhile, Cell apoptosis was detected using fluorescence-activated cell sorting (FACS). Caspase-3 activity was assayed by spectrophotometry. Fibroblast populated collagen lattice (FPCL) model was employed to evaluate the contractility of HSFb. Further, real-time PCR and western blot were used to measure the mRNA and protein expressions of α-SMA in HSFb. In addition, mRNA levels of type I and III procollagen were assayed by quantitative real-time PCR. The results revealed that shRNA targeting SFRP2 significantly promoted the apoptosis of HSFb, while it had no effect on the cell proliferation. Decreased synthesis of a-smooth muscle actin (α-SMA) in HSFb and reduced contraction of fibroblasts in the FPCL model were observed. Quantitative RT-PCR suggested that the mRNAs of type I and III procollagen were significantly downregulated. In conclusion, as a novel anti-apoptosis gene, SPRP2 was present in hypertrophic scars. Importantly, shRNA targeting SFRP2 may provide a new approach to preventing the formation of HS.

The aim of this study was to investigate whether the JAK-STAT pathway participates in the processes of proliferation and differentiation induced by connective tissue growth factor (CTGF) in human hypertrophic scar fibroblasts (hHSF). hHSF were grown as primary cultures, then treated with or without CTGF. Western blotting was used to detect JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6 protein expression in the hHSF at various time points after stimulation with CTGF. Immunofluorescence and the electrophoretic mobility shift assay (EMSA) were used to identify the interacting signalling molecules and to investigate their activation, respectively. After the signalling molecules of interest were selected, a specific inhibitor (STAT1 ASODN) was used to block the JAK-STAT pathway. The MTT assay was used to detect the proliferation of hHSF, and differentiation was assessed by evaluating changes in α-SMA expression by RT-PCR. Based on the results of Western blotting, immunofluorescence and EMSA, proliferation and differentiation were much higher in hHSF treated with CTGF (p

Epidermal stem cells (ESCs) are essential not only for tissue homeostasis but also for skin to respond to insults, but the mechanisms of stem cell regulation are unknown. To investigate the function of Rac1 in ESC development, we introduced either the dominant negative isoform or constitutively the active mutant of Rac1 in cultured human ESCs by using a retroviral vector and then analyzed the consequences. Upon activation, Rac1 increased surface alpha6/beta1 integrin levels and promoted colony forming efficiency of ESCs. Conversely, dominant negative Rac1 caused a progressive reduction in growth rate, an inhibition of adhesiveness and a marked stimulation of terminal differentiation, without any effect on the cell cycle. These results were consistent with the role of Rac1 in determining the fate of ESCs by controlling their exit from the stem cell compartment. Our results reveal a novel biological role for Rac1 and provide new insights into the mechanism regulating ESCs.

The mechanisms of hypertrophic scar formation are not fully understood. We previously screened the differentially expressed genes of human hypertrophic scar tissue and identified P311 gene as upregulated. As the activities of P311 in human fibroblast function are unknown, we examined the distribution of it and the effects of forced expression or silencing of expression of P311. P311 expression was detected in fibroblast-like cells from the hypertrophic scar of burn injury patients but not in peripheral blood mononuclear cells, bone marrow mesenchymal stem cells, epidermal cells or normal skin dermal cells. Transfection of fibroblasts with P311 gene stimulated the expression of alpha-smooth muscle actin (alpha-SMA), TGF-beta1 and alpha1(I) collagen (COL1A1), and enhanced the contraction of fibroblast populated collagen lattices (FPCL). In contrast, interference of fibroblast P311 gene expression decreased the TGF-beta1 mRNA expression and reduced the contraction of fibroblasts in FPCL. These results suggest that P311 may be involved in the pathogenesis of hypertrophic scar via induction of a myofibroblastic phenotype and of functions such as TGF-beta1 expression. P311 could be a novel target for the control of hypertrophic scar development.

CCAAT/enhancer-binding protein β (C/EBPβ), also known as nuclear factor–interleukin-6 (NF-IL6), is a transcription factor that plays an important role in the regulation of growth and differentiation of myeloid and lymphoid cells. Mice deficient in C/EBPβ show impaired generation of B lymphocytes. We show that C/EBPβ regulates transcription factors critical for proliferation and survival in multiple myeloma. Multiple myeloma cell lines and primary multiple myeloma cells strongly expressed C/EBPβ, whereas normal B cells and plasma cells had little or no detectable levels of C/EBPβ. Silencing of C/EBPβ led to down-regulation of transcription factors such as IRF4, XBP1, and BLIMP1 accompanied by a strong inhibition of proliferation. Further, silencing of C/EBPβ led to a complete down-regulation of antiapoptotic B-cell lymphoma 2 (BCL2) expression. In chromatin immunoprecipitation assays, C/EBPβ directly bound to the promoter region of IRF4, BLIMP1, and BCL2. Our data indicate that C/EBPβ is involved in the regulatory network of transcription factors that are critical for plasma cell differentiation and survival. Targeting C/EBPβ may provide a novel therapeutic strategy in the treatment of multiple myeloma.

The mechanisms by which the human T-cell leukemia virus type I (HTLV-I) Tax oncoprotein deregulates cellular signaling for oncogenesis have been extensively studied, but how Tax itself is regulated remains largely unknown. Here we report that Tax was negatively regulated by PDLIM2, which promoted Tax K48-linked polyubiquitination. In addition, PDLIM2 recruited Tax from its functional sites into the nuclear matrix where the polyubiquitinated Tax was degraded by the proteasome. Consistently, PDLIM2 suppressed Tax-mediated signaling activation, cell transformation, and oncogenesis both in vitro and in animal. Notably, PDLIM2 expression was down-regulated in HTLV-I–transformed T cells, and PDLIM2 reconstitution reversed the tumorigenicity of the malignant cells. These studies indicate that the counterbalance between HTLV-I/Tax and PDLIM2 may determine the outcome of HTLV-I infection. These studies also suggest a potential therapeutic strategy for cancers and other diseases associated with HTLV-I infection and/or PDLIM2 deregulation.

Hypertrophic scar (HS) remains a major problem in plastic surgery. In order to explore the regulative effect of phosphatase and tensin homolog (PTEN) on HS, PTEN and AKT expression was detected by reverse transcription PCR, immunohistochemistry and western blot. Adenovirus-mediated PTEN overexpression in cultured hypertrophic scar fibroblasts (HSFBs) and normal skin fibroblasts was also introduced to evaluate its biological function. Our results showed that PTEN expression was significantly decreased in HS whereas p-Akt level was significantly higher in HS compared with normal skin (P < 0.01). Furthermore, we found that adenovirus-mediated PTEN overexpression led to decreased AKT activation, and significantly reduced cell proliferation and collagen synthesis of HSFBs, while increased the apoptosis. Taken together, these data suggest that PTEN inhibits proliferation and function of HSFBs through AKT pathway. Our results reveal a novel biological role for PTEN/AKT pathway in HS and suggest PTEN as a potential therapeutic target for HS treatment.

Fuqiang Pan, Rui Guo, Wenguang Cheng, Linlin Chai, Wenping Wang, Chuan Cao, Shirong LiDepartment of Plastic and Reconstructive Surgery, Southwestern Hospital, Third Military Medical University, Chongqing, People’s Republic of China Background: Accumulating evidence has demonstrated that migration of keratinocytes is critical to wound epithelialization, and defects of this function result in chronic delayed-healing wounds in diabetes mellitus patients, and the migration has been proved to be associated with volume-activated chloride channels. The aim of the study is to investigate the effects of high glucose (HG, 25mM) on ClC-2 chloride channels and cell migration of keratinocytes.Methods: Newborn Sprague Dawley rats were used to isolate and culture the keratinocyte in this study. Immunofluorescence assay, real-time polymerase chain reaction, and Western blot assay were used to examine the expression of ClC-2 protein or mRNA. Scratch wound assay was used to measure the migratory ability of keratinocytes. Transwell cell migration assay was used to measure the invasion and migration of keratinocytes. Recombinant lentivirus vectors were established and transducted to keratinocytes. Whole-cell patch clamp was used to perform the electrophysiological studies.Results: We found that the expression of ClC-2 was significantly inhibited when keratinocytes were exposed to a HG (25mM) medium, accompanied by the decline of volume-activated Cl- current (ICl,vol), migration potential, and phosphorylated PI3K as compared to control group. When knockdown of ClC-2 by RNAi or pretreatment with wortmannin, similar results were observed, including ICl,vol and migration keratinocytes were inhibited.Conclusion: Our study proved that HG inhibited ClC-2 chloride channels and attenuated cell migration of rat keratinocytes via inhibiting PI3K signaling.Keywords: high glucose, keratinocytes, ClC-2, cell migration, PI3K

Introduction: Lenalidomide (LEN) and pomalidomide (POM) are derivatives of thalidomide (IMiD® compounds) and currently used to treat multiple myeloma (MM) and B-cell malignancies. We have shown before that IMiD® compounds shift lineage commitment of CD34+ cells towards myeloid development by affecting critical transcription factors such as GATA1 and PU.1 with concomitant inhibition of cell maturation resulting in anemia and neutropenia. Nonetheless the underlying pathomechanism is still unknown. Recently, IMiD® compounds were shown to bind to cereblon (CRBN) in MM cells, which is the substrate recognition component of cullin-dependent ubiquitin ligase and LEN leads to ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IZKF3 by the CRBN-CRL4 ubiquitin ligase. We investigated here the role of CRBN, IKZF1 and IKZF3 in IMiD®-induced effects on lineage commitment and maturation of CD34+ cells. Methods and Results: By western blot analysis we found that CRBN and IKZF1, but not IKZF3 are expressed in CD34+ cells. Treatment of CD34+ cells with LEN and POM for only 1h almost completely decreased the expression of IKZF1 without affecting CRBN protein expression. By using a thalidomide analog bead assay, we found that IMiD® compounds directly bind CRBN in CD34+ cells. In contrast to our protein studies, IKZF1-mRNA level was not altered in real-time PCR, suggesting that IMiD® compounds regulate IKZF1 at post-transcriptional level. Treatment with proteasome inhibitors MG132, PS341 or MLN4924 which function as cullin-dependent ubiquitin ligase inhibitors blocked LEN and POM induced IKZF1 degradation, confirming that IMiD® compounds induce ubiquitination and subsequent protein degradation of endogenous IKZF1. Next we generated lentiviral constructs to knockdown the expression of CRBN in CD34+ cells. Knockdown of CRBN in CD34+ cells induced resistance to POM-induced IKZF1 downregulation and subsequently reversed the POM-induced lineage shift in colony-formation assays, suggesting that POM-induced degradation of IKZF1 in HSC requires CRBN.Knowing that PU.1 and GATA-1 are critically involved in the IMID-induced lineage shift in CD34+ cells we demonstrated in chromatin immunoprecipitation assays that IKZF1 binds to promoter regions of PU.1 and GATA-1, suggesting that PU.1 and GATA-1 are direct downstream targets of IKZF1 in CD34+ cells . Conclusion: Our findings show that CRBN and IKZF1 mediate the effects of IMiD® compounds on hematopoietic progenitors. IMiD® compounds promote CRBN dependent degradation of IKZF1 protein in CD34+ cells that subsequently decreases transcription factors such as GATA1 and PU.1 which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Our findings that IMiD® compounds mediate their effects via CRBN and IKZF1 provide for the first time the pathomechanism how LEN and POM affect hematopoiesis and induce neutropenia, thrombocytopenia as well as anemia. Disclosures Lentzsch: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Research Funding.

Introduction:IMiD® compounds such as lenalidomide (LEN) and pomalidomide (POM) represent a novel class of agents in the treatment of multiple myeloma. Increasing concerns about its potential toxicity on stem cells resulting in an increase of secondary malignancies primarily affecting the myeloid cells have been reported. However, the exact mechanism is unclear. MicroRNAs (miRNAs) are emerging as important regulators in stem cell development. Therefore, the present study investigated the effect of IMiD® compounds on stem cell renewal/proliferation and the role of miRNAs involved in this process. Methods and Results: When human CD34+ cells were cultured with POM or DMSO under conditions favoring myeloid development, POM significantly increased the percentage of CD34+cells in S phase in cell cycle analysis with concomitant dramatic decrease of apoptotic cells. This resulted in a strong and significant proliferation of CD34+cells as well as induction of colony formation. Systematic screening of miRNAs expression profile by microarray showed that POM significantly enhanced the expression of miR-125b. Upregulation of miR-125b was further confirmed by realtime PCR. Interestingly, recently miR-125b was shown to function as an oncomir by inducing tumorigenesis in myeloid cells by So et al., Blood 2014. In accordance with that we found that overexpression of miR-125b in CD34+ cells exhibited similar effects as POM on CD34+ cells, such as increased cell number in S-phase, decreased cell apoptosis resulting in significant induction of colony formation and the absolute number of hematopoietic progenitors. Bioinformatics prediction models revealed a potential binding site of miR-125b on 3'UTR of P53. Indeed subsequent experiments showed that P53 was downregulated when miR-125b was overexpressed in CD34+cells or when treated with POM. Further analysis of the P53 down stream targets revealed that POM decreased the expression of PUMA and BAK1 all critical for control of cell proliferation and apoptosis. The POM-induced upregulation of mir-125b was independent of the CRBN/IKZF1 induced protein degradation. Conclusion: Based on our results we postulate that POM upregulates the oncomir miR-125b in CD34+cells. Mir-125b controls proliferation and apoptosis by downregulating P53 and genes involved in the P53 pathway including BAK1 and PUMA. Thus, our findings on the effects of POM on the miR-125b/P53 axle provide for the first time a potential mechanism for the development of secondary hematologic malignancies in patients treated with IMiD® compounds. Disclosures Lentzsch: Celgene: Consultancy, Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that has diverse activities in mediating host defense and immunopathology and is involved in both innate an adaptive immunity. Furthermore, IFN-γ has been shown to be a key regulator of GVHD. STAT1 is the major downstream signal transducer for IFN-γ. IFN-γ has been shown to up-regulate MHC class II expression on macrophages and dendritic cells. In this study, we unexpectedly observed that STAT1-deficient mouse bone marrow-derived dendritic cells (BM-DCs) displayed significantly increased MHC class II, CD86, CD80, CD40 expression compared to wild type BM-DCs upon LPS stimulation. To determine whether these LPS-maturated STAT1-/- BM-DCs are functional, freshly isolated pan-T cells from BALB/c (H2d) mice were stimulated with either irradiated LPS-maturated STAT1+/+ or STAT1-/- BMDCs from 129Sv [H2b] mice at different DC/Responder ratios for 5 days. Pan-T cell proliferation was measured using by 3H-incorporation or in separate assays by CFSE dilution. Both proliferation assays clearly demonstrated that STAT1-/- BM-DCs were significantly more potent stimulators in Mixed Lymphocyte Reaction (MLR) assays compared to STAT1+/+ BM-DCs. Further studies demonstrated that this increased surface MHC II expression was also observed in LPS-stimulated IFNγR-/- BM-DCs or on wild type BM-DCs treated with neutralizing IFNγ Abs but not in LPS-treated IFNα-R-/- BM-DCs. STAT1 deficient DCs have been reported to be defective in IL-12 production upon stimulation with various Toll-like receptors (TLRs). However, we were able to observe that STAT1-/- BM-DCs had increased CD40 expression after LPS maturation, and more importantly, STAT1 deficient BM-DCs had significantly increased IL-12 production upon treatment with CD40-ligand. These data suggest that STAT1 deficiency in LPS-stimulated DCs results not only in increased expression of MHC class II and costimulatory molecules, but also in enhanced CD40-dependent IL-12 production promoting generation of Th1 and Tc1 cell of wildtype responder cells. To test the influence of IFNγ/STAT1 deficiency in host APC on GVHD induction, wild type or STAT1-deficient 129 mice (H2b) underwent allogeneic Bone Marrow Transplantation (BMT) following lethal irradiation (1044 rad) receiving BALB/c spleen cells. 129.STAT1-/- recipients had significantly accelerated GVHD mortality (MST 8 days vs. 5 days, log rank test p=0.004) compared with wild type recipients. In contrast, STAT1-deficient syngeneic controls did not show enhanced mortality or morbidity ruling out that the observed accelerated mortality following allogeneic BMT was due to conditioning toxicity. On day+4 post-BMT animals were sacrificed and splenocytes were analyzed by flow cytometry. We could observe a significantly enhanced expansion and activation of donor CD4 and CD8 T cells in STAT1-/- recipients. Importantly, the increased MHC II expression was confirmed in host CD11b+ and CD11c+ cell populations. To clarify that STAT1 deficiency in host APCs do contribute to increased GVHD induction, we generated STAT1-/-→STAT1+/+, and STAT1+/+→STAT1+/+ radiation chimeric mice as recipients so that only the hematopoietic cells were STAT1 deficient. We again observed significantly increased GVHD mortality and increased activation of fully MHC-mismatched donor T cells in STAT1-/-→STAT1+/+ recipients following allogeneic BMT and GVHD induction. Similar results i.e. increased GVHD mortality, increased donor cell activation and increased MHC II expression on host CD11c+, B220+ cells were also found in IFNγR-/- recipient mice when compared with control mice following full MHC-mismatched allogeneic BMT. In summary our data reveal previously unknown effects of IFNγ/STAT1 signaling on APCs function suggesting that LPS-driven maturation of APC’s is negatively regulated by IFNγ/STAT1 leading to enhanced expression of MHC class II, costimulatory molecules and responsiveness to CD40-ligation and subsequent IL-12 production in the absence of STAT1. Furthermore, our results explain the accelerated GVHD induction and demonstrate that IFN-γ/STAT1 signaling in host APC mitigates the development of GVHD. Disclosures: Lentzsch: Celgene: Research Funding.

Introduction Overexpression and/or activation of eukaryotic initiation factor 4E (eIF4E) is critical for oncogenic protein synthesis. Mutations in genes related to mRNA translation are involved in the pathogenesis of multiple myeloma (Chapman, Lawrence et al. 2011). Recently, we found that MM cells express high levels of eIF4E protein compared to normal plasma cells and overexpression of eIF4E induces transcription factors such as c-myc critical for the growth of multiple myeloma cells (Li, Fu et al. 2011,2012). The understanding of the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. Here we show the critical role of eIF4E driven protein synthesis by using an inducible knockdown system to silence eIF4E gene expression and confirm the critical role of eIF4E in multiple myeloma growth in vivo and in vitro. Methods and Results We stably infected U266, RPMI-8226, IM-9 and MM.1S cells with a robust inducible single-lentiviral knockdown vector pLKO-Tet-On containing either control non-targeting shRNA or eIF4E targeting shRNA sequences. Doxycycline-induced eIF4E shRNA expression resulted in significant decrease of eIF4E mRNA and protein in eIF4E-shRNA but not the control shRNA infected MM cells. To determine the effects of eIF4E knockdown on MM cell growth and viability, stably transfected cell lines were grown in the presence or absence of doxycycline. Silencing of eIF4E by doxycycline induction of eIF4E shRNA in RPMI-8226 cells significantly inhibited (>72%,P Conclusion Here we show that eIF4E, a key player in the translational machinery, promotes multiple myeloma cell growth. We found that high eIF4E expression is indispensable for the growth of MM cells both in vitro and in vivo. Silencing of eIF4E decreases protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation, and resulted in tumor inhibition. Our study indicated that targeting transcriptional initiating factor eIF4E may represent a novel therapeutic strategy for MM treatment. Disclosures: Schecter: Seattle Genetics: Honoraria, Research Funding. Lentzsch:Celgene: Research Funding.

Abstract 4025 Background: MM cells produce several osteoclast-activating factors, which result in highly activated osteoclasts and cause dysregulated bone remodeling with excessive bone resorption. Our previous data showed that matrix metalloproteinase 13 (MMP13) is highly expressed in MM cells, and co-culture with stromal cells further increased MMP13 levels. Most importantly, exogenous MMP13 increased OCL fusion and bone resorption activity induced by nuclear factor kappa-B ligand (RANKL). Here, we further addressed the mechanism of MMP13 upregulation in MM and its role in MM-related bone disease in vivo. Methods and Results: Based upon the previous results that IL-6 neutralizing antibody blocks MMP13 upregulation in MM cells co-cultured with stromal cells, we further investigated the role of IL-6 on MMP13 induction in MM cells. Human RPMI8266 MM cells were serum-starved for 24 hours, then treated with IL-6 for up to 96 hours. RT-PCR and western blotting showed that MMP13 expression and secretion by MM cells were upregulated after 24h and prolonged through 96h. AP-1 binding sites were identified in the MMP13 promoter, and we found that IL-6 induced upregulation of the AP-1 members c-Jun and c-fos in MM cells by RT-PCR and western blotting, which correlated with MMP13 induction. To address the role of heightened MMP13 expression/secretion of MM on OCL formation and development of lytic lesions, we silenced MMP13 expression in MM cells by lentiviral mediated shRNA transduction. 5TGM1 mouse MM cells were infected with the pKLO.1-puro-vector (EV) or pKLO.1-puro-sh-MMP13 (MMP13 Knock down [KD]) lentivirus particles and cells were selected by puromycin. MMP13 KD was confirmed by RT-PCR and western blotting. To investigate the effects of MMP13 KD on OCL formation, we co-cultured mouse bone marrow cells (BMC) with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells using a transwell system to permit only soluble molecule exchange. Co-culture with 5TGM1-EV MM cells significantly (p Conclusion: Our results demonstrate that IL-6-induced high level of MMP13 in MM cells is essential for multiple myeloma tumor growth, OCL induction and development of lytic bone lesions. Disclosures: Lentzsch: Celgene: Consultancy, Research Funding.

Abstract 2908 Introduction: The eukaryotic initiation factor 4E (eIF4E) is a critical regulator in protein synthesis. It has been shown that overexpression and/or activation of eIF4E is critical for oncogenic protein synthesis. The precise role of protein translation in multiple myeloma (MM) is less clear. Recently it has been shown that eIF4E protein levels are higher in primary CD138+ MM cells than in normal plasma cells (Li, Jin et al. 2011) and that mutations in genes related to mRNA translation are involved in the pathogenesis of multiple (Chapman, Lawrence et al. 2011). Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. In this study, we analyzed the effects of introduction of ectopic eIF4E in MM cell lines compared with their parent cells in vitro and in vivo. Results: To examine the effect of overexpressed eIF4E in MM, we transduced MM cell lines with lentiviral particles encoding human eIF4E with GFP as selection marker. Introduction of ectopic eIF4E significantly increased critical factors for myeloma cell growth such as myc, cyclin D1, C/EBP beta and IRF4 as detected by western blotting. Overexpression of eIF4E resulted in a significant (p Conclusion: Here we show that eIF4E, a key player in translational machinery, promotes multiple myeloma cell growth both in vitro and in vivo. When eIF4E is overexpressed, it enhances protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation. Disclosures: Lentzsch: Celgene: Consultancy, Research Funding.

Abstract 1853 Methods: The translation initiation factor eIF4E is central to protein synthesis in general, and overexpression and/or activation of eIF4E is associated with a malignant phenotype by regulating oncogenic protein translation. Several previous publications indicate that aberrant control of protein synthesis contributes to lymphoma genesis but the exact role of protein translation in multiple myeloma (MM) is less clear. Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. The goal of this study was to determine the role and regulation of eIF4E, as well as the effects of protein translation controlling drugs in MM. Results: By western blot analysis as well as RT-PCR we found that eIF4E protein and mRNA levels are significantly elevated (up to 20 fold) in MM cell lines (H929, RPMI-8226, MM.1S and OPM2) and primary myeloma cells compared to normal plasma cells. Silencing of eIF4E gene expression in RPMI-8226 MM cells by a stable and inducible shRNA system significantly decreased viability of myeloma cells (by ∼ 43%) but not of HEK 293 suggesting a higher dependency of MM cells to protein translation. Next we evaluated different drugs including pomalidomide, rapamycin, pp242, 4EGI-1 and ribavirin, that are known to inhibit protein synthesis for their effects on protein translation in MM. By m7GTP pull down assays we evaluated the effects of the different drugs on eIF4E expression and activity. Rapamycin blocked the phosphorylation of 4EBP1 and eIF4E release, and subsequently inhibited eIF4G binding. The compound 4EGI-1 decreased the interaction between eIF4E and eIF4G. Pomalidomide decreased eIF4E protein expression. All drugs inhibited MM cell DNA synthesis measured by 3H-Thymidine incorporation. Treatment with pomalidomide (10uM), rapamycin (40nM), pp242 (10uM), 4EGI1 (50uM) or ribavirin (50uM) for 48h significantly decreased (p Conclusion: Here we show that eIF4E, a key player in translational control, is highly expressed in MM cells and critical for MM growth and survival. Therefore our study helps to understand the function and regulatory mechanism of eIF4E in MM. Further the evaluation of drugs targeting protein translation provides the basis for the optimization of current MM treatment or to open up new strategies such as targeting protein translation in future MM therapy. Disclosures: Lentzsch: Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.

Abstract 2973 We have recently shown that loss of STAT1 signaling in donor T cells significantly reduces acute GVHD in both MHC-mismatched and mHA-mismatched BMT and is associated with increased expansion of Treg cells. In this study we further addressed the role of STAT1 in GVL reactions and the underlying mechanisms. We first sought to delineate the role of STAT1 in the generation of CD8+ CTL against host-type spleen cells and A20 lymphoma targets using standard CML assays. Whole spleen cells (SPCs) or CD8+ selected STAT1+/+ or STAT1−/− cells from 129Sv mice (H2b) were stimulated for 5 days with irradiated BALB/c (H2d) spleen cells or A20 cells and then assessed for CTL activity against 51Cr-labelled target cells. STAT1-deficient SPCs appeared to have reduced CTL activity against host SPCs and host type A20 lymphoma cells compared to STAT1+/+ splenocytes. In contrast, isolated STAT1-deficient CD8+ cells had significantly increased CTL activity against both host-type targets and A20 cells. We then assessed the role of STAT1 in mediating GVL effects in vivo using a fully MHC-mismatched (129 [H2b]→BALB/c [H2d]) strain combination and the A20 lymphoma model. Lethally irradiated (800rad) BALB/c mice received T cell-depleted (TCD) wildtype 129 bone marrow cells (BMCs, 5×10E6) plus pan-T cells (5×10E5) from either 129.STAT1−/− or 129.STAT1+/+ donors. In addition, recipients were injected with 5×10E5 luciferase-expressing A20 lymphoma cells (A20-luc) on day 0. As expected animals receiving STAT1-deficient T cells showed significantly reduced GVHD-induced mortality (mean survival time [MST] not reached versus 11 days, p Disclosures: Lentzsch: Celgene: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; Centocor Ortho Biotech: Research Funding.