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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.

Aim: The role of plasma heat shock protein 90 alpha (HSP90α) in colorectal cancer patients remains unclear. This study aimed to evaluate the relationship between HSP90α and the occurrence and development of colorectal cancer through diagnosis and prognosis value.Methods: 635 colorectal cancer patients and 295 healthy controls were recruited. The HSP90α was measured by using the ELISA kit in all objects and the immune cells and common biomarkers as CEA, AFP, CA125, CA153 and CA199 were measured in all colorectal cancer patients. The relationship between plasma HSP90α with clinical features, common tumor markers and immune cells were also conducted. The survival analysis endpoint was progression-free survival (PFS).Results: The levels of plasma HSP90α were significantly higher in colorectal cancer patients compared to healthy controls [51.4 (ng/ml) vs. 43.7 (ng/ml), p < 0.001]. In additional, the levels of plasma HSP90α were associated with gender and disease progress as stage, lymphatic and distant metastasis. Furthermore, plasma HSP90α was closed correlation with CEA, CA125, CA199 and percentage of B cells. However, the initial expression level of plasma HSP90α failed to show a prognostic value for progression-free survival in colorectal cancer.Conclusion: The plasma Hsp90α was remarkable higher in colorectal cancer and correlated with common tumor biomarkers and immune cells. Plasma Hsp90α levels were associated with disease progress but a poor diagnosis performance and also failed to show a prognostic value in colorectal cancer.

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.

Background Idiopathic pulmonary fibrosis (IPF) is a progressive disease resulting in respiratory failure with no efficient treatment options. We investigated the protective effect of RS4651 on pulmonary fibrosis in mice and the mechanism. Methods Intratracheal injection of bleomycin (BLM) was used to induce pulmonary fibrosis in mice. RS4561 was administered intraperitoneally at different doses. Histopathological changes were observed. The level of alpha-smooth muscle actin (α-SMA) were also tested. In vitro, the proliferation and migratory effects of RS4651 treatment on MRC-5 cells pre-treated with transforming growth factor (TGF-β1) were examined. RNA-sequencing was used to detect differentially expressed target genes. Then, the expression of α-SMA, pSMAD2 and SMAD7 were analysed during RS4651 treatment of MRC-5 cells with or without silencing by SMAD7 siRNA. Results Histopathological staining results showed decreased collagen deposition in RS4651 administered mice. Additionally, a lower level of α-SMA was also observed compared to the BLM group. The results of in vitro studies confirmed that RS4651 can inhibit the proliferation and migration, as well as α-SMA and pSMAD2 expression in MRC-5 cells treated with TGF-β1. RNA-sequencing data identified the target gene SMAD7. We found that RS4651 could upregulate SMAD7 expression and inhibit the proliferation and migration of MRC-5 cells via SMAD7, and RS4651 inhibition of α-SMA and pSMAD2 expression was blocked in SMAD7-siRNA MRC-5 cells. In vivo studies further confirmed that RS4651 could upregulate SMAD7 expression in BLM-induced lung fibrosis in mice. Conclusions Our data suggest that RS4651 alleviates BLM-induced pulmonary fibrosis in mice by inhibiting the TGF-β1/SMAD signalling pathway.

Background Mesenchymal stem cell-based acellular therapies have been widely exploited in managing hypertrophic scars. However, low maintenance dose and transitory therapeutic effects during topical medication remain a thorny issue. Herein, this study aimed to optimize the curative effect of adipose-derived stem cell conditioned medium (ADSC-CM) in the prevention of hypertrophic scarring. Methods In the present study, ADSC-CM was concentrated via the freeze-drying procedure. The efficacy of different dose groups (CM, CM5, CM10) was conducted on the proliferation, apoptosis, and α-smooth muscle actin (α-SMA) expression of human keloid fibroblasts (HKFs) in vitro. Incorporation of adipose-derived stem cell concentrated conditioned medium (ADSCC-CM) into polysaccharide hydrogel was investigated in rabbit ear, in vivo. Haematoxylin-eosin (H&E) and Masson’s trichrome staining were performed for the evaluation of scar hyperplasia. Results We noted that ADSCC-CM could downregulate the α-SMA expression of HKFs in a dose-dependent manner. In the rabbit ear model, the scar hyperplasia in the medium-dose group (CM5) and high-dose group (CM10) was inhibited with reduced scar elevation index (SEI) under 4 months of observation. It is noteworthy that the union of CM5 and polysaccharide hydrogel (CM5+H) yielded the best preventive effect on scar hyperplasia. Briefly, melanin, height, vascularity, and pliability in the CM5+H group were better than those of the control group. Collagen was evenly distributed, and skin appendages could be regenerated. Conclusions Altogether, ADSCC-CM can downregulate the expression of α-SMA due to its anti-fibrosis effect and promote the rearrangement of collagen fibres, which is integral to scar precaution. The in situ cross bonding of ADSCC-CM and polysaccharide hydrogel could remarkably enhance the therapeutic outcomes in inhibiting scar proliferation. Hence, the alliance of ADSCC-CM and hydrogel may become a potential alternative in hypertrophic scar prophylaxis.

OBJECTIVE. This study aims to assess correlations of the time from symptom onset to diagnosis and treatment with the time to disease resolution and CT scores as based on findings from sequential chest CT examinations. MATERIALS AND METHODS. Thirty patients with coronavirus disease (COVID-19) confirmed by reverse transcription-polymerase chain reaction analysis underwent chest CT examinations. Five patients who did not have positive CT findings or who had not yet fulfilled criteria for discharge from the hospital were excluded. CT scores were determined according to CT findings and lung involvement. The time from symptom onset to diagnosis and treatment was recorded for each patient, and on the basis of this information, patients with COVID-19 were divided into group 1 (patients for whom this interval was ≤ 3 days) and group 2 (those for whom this interval was > 3 days). The CT scores for each group were fitted using a Lorentzian line-shape curve to show the variation tendency during treatment. The differences in age, sex, and last CT scores determined before discharge between the two groups were analyzed, and correlations of the time from symptom onset to diagnosis and treatment with the time to disease resolution as well as with the highest CT score also underwent statistical analysis. RESULTS. A total of 25 subjects were enrolled in the study. The fitted tendency curves for group 1 and group 2 were significantly different, with peak points showing that the estimated highest CT score was 10 and 16 for each group, respectively, and the time to disease resolution was 6 and 13 days, respectively. The Mann-Whitney test showed that the last CT scores were lower for group 1 than for group 2 (p = 0.025), although the chi-square test found no difference in age and sex between the groups. The time from symptom onset to diagnosis and treatment had a positive correlation with the time to disease resolution (r = 0.93; p = 0.000) as well as with the highest CT score (r = 0.83; p = 0.006). CONCLUSION. Timely diagnosis and treatment are key to providing a better prognosis for patients with COVID-19.

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.

Introduction Multiple myeloma (MM)-induced bone disease remains one of its most devastating complications, caused by increased bone resorption by overactivated osteoclasts coupled with impaired bone formation. MM cells produce osteoclast-activating factors that induce osteoclast activation and extensive bone resorption. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells (Fu J etc. JCI. 2016). We also identified that the checkpoint inhibitor, programmed death-1 homolog (PD-1H/VISTA), serves as the MMP-13 receptor in osteoclasts and mediates MMP-13-dependent osteoclastogenic function which is largely blocked in Pd-1h -/-osteoclasts (Fu J etc. ASH 2019, 2020). While the inhibitory role of PD-1H/VISTA in T-cells has recently been described (ElTanbouly MA etc. Science 2020), its cellular binding proteins remain unclear, and its role in osteoclast activation andMM bone disease have not been addressed. Methods and Results To identify its interacting proteins, PD-1H-His 6 recombinant protein was expressed in mouse bone marrow mononuclear cells, the associated proteins pulled down by Ni-NTA agarose beads from cell lysates and identified by mass spectrometry. Functional annotation charting of the 75 proteins enriched in PD-1H pull-down samples (with signal ratio of PD-1H-His 6 pull-down vs control >2) indicated that almost 30% of the interacting targets were either cytoskeletal or cytoskeleton-associated proteins. Given that the F-actin cytoskeleton undergoes dynamic reorganization during osteoclast differentiation and plays critical roles in bone resorption, we further addressed the role of PD-1H in F-actin cytoskeleton regulation. Initially, osteoclasts form F-actin-rich adhesive structures, termed podosomes. At later stages, podosomes collectively rearrange into clusters and rings and finally into sealing belts to mediate osteoclast spreading, migration and bone resorption (Teitelbaum SL. Ann N Y Acad Sci. 2011). By confocal immunofluorescence microscopy, we found that PD-1H co-localized with F-actin podosome clusters, rings and sealing belts during osteoclast differentiation (Figure 1A). The functional role of PD-1H in F-actin cytoskeleton reorganization was addressed using Pd-1h -/- osteoclast wherein Pd-1h knockout lead to the disruption of podosome clusters at early stages relative to WT controls, while at later stages, Pd-1h -/-osteoclasts exhibited significantly fewer F-actin rings and belts (Figure 1B). Further, binding of MMP-13 to PD-1H increased the number of osteoclasts forming F-actin rings and belts, as well as the size of F-actin belts, which was blocked in Pd-1h -/- osteoclasts. To determine the role of PD-1H in the development of myeloma-induced lytic bone lesions, 5TGM1 myeloma cells were bilaterally intratibially injected into Pd-1h wtRag2 -/- or Pd-1h -/-Rag2 -/- mice (n=10) to induce lytic bone lesions. Three weeks following intratibial 5TGM1 injection, tibiae were harvested for micro-computed tomography. Subsequent quantitative histomorphology analyses of the trabecular and cortical bones confirmed that the knockout of Pd-1h reduced MM-induced bone destruction with significantly less decrease in trabecular bone volume (Tb. [BV/TV]), trabecular bone number (Tb. N.), trabecular bone thickness (Tb. Th.), as well as less increase in trabecular bone spacing (Tb. Sp.) and bone specific surface (Tb. [BS/BV]) compared to Pd-1h wtRag2 -/- mice. Similar effects were observed in cortical bone with less decrease in cortical bone thickness (CT. Th.), cortical bone area fraction (CT. [BA/TA]), and cortical tissue mineral density (CT. TMD) in 5TGM1 bearing Pd-1h -/-Rag2 -/- mice vs Pd-1h wtRag2 -/- mice (Table 1). Conclusions Taken together, our study, for the first time, reveals the novel role of checkpoint inhibitor, PD-1H/VISTA, in osteoclasts and myeloma bone disease. PD-1H associates with cytoskeleton proteins and regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Further, PD-1H mediates MMP-13-induced osteoclast fusion, F-actin belts formation, and osteoclast activation. Pd-1h -/-in recipient mice significantly impairs MM-induced bone loss, demonstrating that PD-1H/VISTA plays a critical role in MM bone disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

Background Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues, and neurological deficits. MM cells secret pro-osteoclastogenic factors which lead to osteoclast (OCL) activation. Our previous work showed that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor highly secreted by MM cells and induces OCL fusion and bone resorption independently of its proteolytic activity (JCI 2016). We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed on OCLs and mediates MMP-13 induced OCL fusion and bone resorption activity (ASH 2019). Bone resorption activity is significantly impaired in Pd-1h-/- OCLs in vitro. However, the function of PD-1H in MM bone disease has not been defined. Methods The role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model. Pd-1h-/-Rag2-/- mice were generated by crossbreeding Pd-1h-/- with Rag2-/- mice. Firefly luciferase-expressing 5TGM1 cells were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=10). 3 weeks later, tibiae were harvested for quantitative micro-CT followed by histological analysis. Results Morphological analyses of trabecular and cortical bones confirmed that Pd-1h-/- recipient mice exhibited significantly less 5TGM1-induced bone loss (P Conclusions Taken together, our study, for the first time, reveals that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation, and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/- mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment.

Introduction Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues and neurological deficits. MM cells produce osteoclast-activating factors that induce osteoclast activation, thereby leading to excessive bone resorption and lytic bone lesions1. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption via a mechanism independent of its proteolytic activity2. We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed in osteoclasts at high levels3. Binding of MMP-13 to PD-1H/VISTA induces osteoclast fusion and bone resorption activity whereas knockdown or knockout of PD-1H/VISTA largely block MMP-13 mediated effects on osteoclasts3. However, the function of PD-1H inMM bone disease in vitro or in vivo has not been previously defined. Methods and Results To confirm the role of PD-1H in MMP-13 induced bone disease in MM, we first conducted MM-osteoclast trans-well co-culture assay using murine MM cell line, 5TGM1 cells, and bone marrow mononuclear cells from Pd-1h-/- or wild type (WT) mice. 5TGM1 control cells or MMP-13 knockdown 5TGM1 cells were seeded in the upper wells of the transwell plates; while WT or Pd-1h-/- bone marrow mononuclear cells were seeded in the lower wells and cultured for osteoclast differentiation assessed by TRAP staining. Results show that 5TGM1 induced differentiation of WT osteoclasts with significantly increased osteoclast size and nuclei number/osteoclast. Consistent with our previous results2, MMP-13 knockdown blocked the 5TGM1 MM cells-induced activation of WT osteoclasts. In contrast, neither 5TGM1 MM cells nor MMP-13 knockdown cells had significant effects on Pd-1h-/- osteoclasts. Hence, knockout of Pd-1h abrogated MMP-13 mediated MM induction of osteoclasts, indicating that MMP-13/PD-1H interactions are critically involved in MM-induced osteoclast activation. The in vivo role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model2. For this purpose, Pd-1h-/-Rag2-/- mice were generated by crossbreeding C57BL/6 Pd-1h-/- with C57BL/6 Rag2-/- mice. 3x105 firefly luciferase expressing 5TGM1 cells (5TGM1-luc) were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=5). Tumor progression was monitored by weekly bioluminescence imaging (BLI). 3 weeks after tumor inoculation, tibiae were harvested for quantitative micro-CT, followed by histological analysis. Histological staining showed that intratibial injection of 5TGM1-luc MM cells induced extensive lytic lesions and trabecular bone loss in Rag2-/- mice. In contrast, in Pd-1h-/-Rag2-/- mice,the bone structure was maintained with markedly less bone loss. Morphological analyses of trabecular bone across proximal tibiae further indicated that in Rag2-/- mice, 5TGM1 induced significant changes in bone microarchitecture, with decreased bone volume fraction (bone volume/tissue volume), connective density, trabecular bone numbers, and trabecular bone thickness, as well as increased trabecular bone spacing (Table 1). In contrast, in Pd-1h-/-Rag2-/- mice, 5TGM1 failed to induce significant loss of trabecular bone, confirming the critical role of PD-1H in MM induced bone disease in vivo. Conclusions Taken together, our study, for the first time, reveal that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/-mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment. References 1. Marino S, Petrusca DN, Roodman GD. Br J Pharmacol. 2019;10.1111/bph.14889. 2. Fu J, Li S, Feng R, et al. J Clin Invest. 2016;126(5):1759-1772. 3. Fu J, Li S, Yang C, et al. Blood. 2019; 134 (Supplement_1): 3072. Disclosures Lentzsch: Caelum Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Celularity: Consultancy, Other; Magenta: Current equity holder in private company; Karyopharm: Research Funding; Mesoblast: Divested equity in a private or publicly-traded company in the past 24 months.

8027 Background: Activating mutations of the RAS genes NRAS, KRAS, and HRAS ( RASmut) occur in up to 50% of MM and portend poor survival and high recurrence rates. MM cells with RASmut are susceptible to inhibition of germinal center kinase (GCK), resulting in IKAROS degradation independent of cereblon (CRBN). Selinexor is a selective inhibitor of nuclear export (SINE) compound that can induce IKAROS degradation through CRBN-independent pathways to overcome immunomodulatory drug resistance. We explored the benefit of selinexor treatment for pts with RASmut MM. Methods: In the randomized BOSTON study, pts with MM after 1-3 therapies received weekly XVd or twice weekly Vd. In the single-arm STORM study pts with penta-treated, triple class refractory MM were treated with twice weekly Xd. Both treatment regimens are now FDA approved. Mutations were assessed post-hoc by exome sequencing of 119 and 52 pts from BOSTON and STORM, respectively. Pts were considered RASmut if their MM had NRAS, KRAS or HRAS mutations in codons 12, 13 or 61. Results: There were 54 pts (45%) with RASmut in BOSTON (XVd = 26, Vd = 28), and 17 (33%) in STORM. In BOSTON, pts with RASmut MM treated with XVd had significantly longer progression-free survival (PFS) than those treated with Vd (median [med] = 12.9 vs 6.7 months [mo], hazard ratio [HR] = 0.48 [95% CI 0.24-0.97], p = 0.039). For pts treated with Vd, those with RASmut had significantly shorter overall survival (OS) compared to RASwild-type (WT) (med = 16.8 mo vs not reached [NR], HR = 2.87 [95% CI 1.03-7.99], p = 0.035). PFS was not significantly different (med = 6.74 vs 9.82 mo, HR = 1.64 [95% CI 0.88-3.07], p = 0.122). Amongst pts on XVd, there was no difference in survival between RASmut and RASWT pts (PFS: med = 12.8 vs 12.9 mo, HR = 1.08 [95% CI 0.52-2.26], p = 0.83; OS: med = NR vs NR, HR = 0.94 [95% CI 0.36-2.45], p = 0.91). In STORM, pts with RASmut had shorter OS compared to RASwt pts (med = 6.1 vs NR, HR = 2.05 [95% CI 1.22-5.19], p = 0.010). Preliminary studies to explore the mechanisms of action related to RASmut MM sensitivity to XVd demonstrated that selinexor treatment in vitro leads to downregulation of GCK. Conclusions: Despite typically having the worst outcomes, pts with RASmut MM had a similar benefit from XVd as RASWT MM, showing that the XVd combination can overcome RASmut. Mechanistically, selinexor induced down regulation of GCK and enhanced killing of RASmut MM cells. With a manageable safety profile, the XVd regimen could provide a viable treatment option to improve survival of pts with MM with RAS mutations. Clinical trial information: NCT03110562. [Table: see text]

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.

Introduction: RAS oncogenes are the most frequently mutated gene family in human cancers. 50% of newly diagnosed multiple myeloma patients carry a RAS/MAPK pathway mutation, with a rising percentage in the relapsed situation1. Thus, targeting RAS mutations in multiple myeloma will increase therapeutic efficiency and potentially overcome drug-resistance. Unfortunately, RAS mutations have been considered "undruggable" due to a lack of traditional small molecule binding pockets on the proteins. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK), also named mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), is an upstream activator in the MAPK pathway. Indeed, we recently discovered the critical role of GCK in RAS mutated (RASmut) MM cell survival and growth. GCK knockdown in RASmut MM cells induced MM cell growth inhibition both in vitro and in vivo. However, the detailed mechanism is yet to be defined. Methods and Results: Our previous data showed that GCK knockdown induces MM cell growth inhibition, associated with the blockage of MKK4/7-JNK phosphorylation and the downregulation of critical transcriptional factors (TFs) including IKZF1/3, BCL-6, and c-MYC proteins. To confirm that GCK knockdown downregulates IKZF1/3 etc at protein level but not mRNA level, we conducted real-time PCR on GCK knockdown MM cells and compared the expression of GCK and TFs to the empty vector (EV) infected MM cells. Results showed that shRNA induced GCK silencing only led to the significantly decreased GCK mRNA, however, did not affect IKZF1 and c-MYC expressions at mRNA level. Consistent with the effects of GCK knockdown, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 proteins, inhibited MM cell proliferation and induced cell apoptosis. IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Importantly, our data showed that IMiDs-resistant RPMI-8226 MM cells have high expression of GCK. GCK knockdown and inhibition induced IKZF1 downregulation, triggered growth inhibition and cell apoptosis in RPMI-8226 cells, suggesting that GCK regulates IKZF1 degradation via a CRBN-independent mechanism. To confirm this hypothesis, we silenced CRBN in N-Rasmut H929 MM cells by shRNA lentiviral infection and examined the response to IMiDs and GCK inhibitor. CRBN knockdown was confirmed by western blotting. CRBN silencing in H929 cells resulted in lenalidomide (LEN) resistance, evidenced by the WTS proliferation assay. In contrast, CRBN silencing failed to rescue N-Rasmut H929 MM cells from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN. Conclusion: Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RASmut MM cells. Importantly, GCK inhibitor downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RASmut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance. GCK inhibitors may represent a novel therapy for the treatment of RASmut MM patients, especially those who are resistant to IMiDs as well as with refractory or relapsed MM. References 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). Disclosures Marcireau: Sanofi: Current Employment. Lentzsch:Karyopharm: Research Funding; Mesoblast: Divested equity in a private or publicly-traded company in the past 24 months; Janssen: Consultancy; Sorrento: Consultancy; Caelum Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celularity: Consultancy; Magenta: Current equity holder in private company; Sanofi: Research Funding.

Keloids are fibroproliferative disorders characterized by the overabundant deposition of extracellular matrix (ECM), especially collagen and overgrowth of scar tissue in response to cutaneous injury. In this study, we isolated a selenium (Se)-containing polysaccharide (Se-ZGTP-I) from Ziyang green tea and explored its potential therapeutic effects on keloid fibroblasts formation. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and annexin V/propidium iodide (PI) staining assays demonstrated that Se-ZGTP-I or neuron-glia 2 (NG2) short hairpin RNA (shRNA) significantly inhibited proliferation of human keloid fibroblasts via induction of apoptosis. Besides, the activation of caspase-3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP) were observed in keloid fibroblasts following Se-ZGTP-I (200 and 400 μg/ml) or NG2 shRNA treatment. Moreover, Western blotting analysis showed that treatment of keloid fibroblasts with Se-ZGTP-I (200 and 400 μg/ml) or NG2 shRNA resulted in an increase of pro-apoptotic protein Bax expression and a decrease in expression levels of anti-apoptotic protein Bcl-2 and NG2. In addition, type I collagen biosynthesis and protein expression in keloid fibroblasts following TGF-β1 stimulation were decreased by Se-ZGTP-I (200 and 400 μg/ml) or NG2 shRNA management. Current findings imply that Se-ZGTP-I has a therapeutic potential to intervene and prevent keloid formation and other fibrotic diseases.

Introduction Multiple myeloma (MM) bone disease is characterized by the development of osteolytic bone lesions due to the over-activation of osteoclast and inhibition of osteoblast cells. MM cells secret pro-osteoclastogenic factors which lead to osteoclast (OCL) activation. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor which is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption by triggering the ERK1/2-dependent up-regulation of the cell fusogen, DC-STAMP. This process operates independently of the MMP-13 proteolytic activity. The fact that MMP-13 regulates OCL signaling via a proteolytic independent mechanism suggests that MMP-13 functions as a paracrine message protein mediating the crosstalk between MM cells and OCL. However, the mechanism is yet to be identified. Methods and Results To screen for MMP-13 cellular binding proteins/receptors, we performed a MMP-13 pull-down assay wherein recombinant MMP-13-His6 was incubated with mouse mononuclear bone marrow cells (BMCs) lysates and Ni-NTA magnetic beads were used to pull-down MMP-13- associated proteins. Following mass spectral analysis, programmed death-1 homolog (PD-1H) was identified as a major MMP-13-binding protein. PD-1H, also known as V-domain Ig suppressor of T cell activation (VISTA), is a critical negative checkpoint regulator expressed on myeloid cells and involved in immune responses. Binding of MMP-13 and PD-1H was confirmed by co-expressing both proteins in HEK293 cells and subsequent co-immunoprecipitation assay. Further, following PD-1H expression in HEK293 cells, an MMP-13-GFP fusion fluorescence protein docked to the cell surface where mutagenesis studies demonstrated that the PD-1H extracellular domain (ECD) mediates the specific binding interaction. Western blot and immunohistochemistry revealed that PD-1H was highly expressed in mononuclear BMCs, pre-OCL and mature OCL. ShRNA-mediated knockdown of PD-1H in mouse mononuclear BMCs blocked the ability of MMP-13 to induce osteoclast fusion and activation. These results were further confirmed by using BMCs from Pd-1h-/- mice and WT littermates for in vitro osteoclast differentiation. While MMP-13 induced WT OCL fusion and activation, these effects were completely blocked in Pd-1h-/-OCLs in tandem with a loss in MMP-13 induced ERK1/2 phosphorylation, NFATc1 and DC-STAMP upregulation. Conclusions Taken together, our study, for the first time, revealed that checkpoint inhibitor PD-1H is highly expressed in pre- and mature osteoclast. More importantly, we identified PD-1H as the critical receptor for MMP-13 in OCL, thereby mediating MMP-13-induced OCL fusion, activation and bone resorption. Hence, MMP-13 as a novel PD-1H ligand might not only induce bone disease, but also play a potential role in the regulation of T-cell activity in MM. As such, targeting MMP-13 and PD-1H interactions may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment. Disclosures Lentzsch: 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; Janssen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Proclara: Consultancy; Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Background To compare the clinical characteristics of Crohn’s disease (CD) and primary intestinal lymphoma (PIL), to find out valuable, relevant indicators for differential diagnosis. Methods The clinical, endoscopic and pathological data of 67 cases (54 cases of Crohn’s disease and 13 cases of primary intestinal lymphoma) admitted by the PLA Army General Hospital in the last seven years were analysed retrospectively. Results The average age of CD was lower than that of PIL, but the course of CD was longer than that of PIL (p Abdominal pain, oral ulcers, perianal lesions, history of surgery (appendectomy or intestinal resection), and multi-site lesions were more common in CD than in PIL (p The biopsy diagnosis rate of PIL was 92.3%, while 85.1% of CD’s pathological manifestations were non-specific inflammation. So the biopsy diagnosis rate of PIL was significantly higher than that of CD (p Two patients were initially diagnosed with CD based on the clinical comprehensive analysis. The clinical symptoms disappeared, and the colonoscopy achieved mucosal healing after treatment. But the patients‘ symptoms repeated, and they were eventually diagnosed as PIL. Conclusions The clinical and endoscopic characteristics of Crohn’s disease (CD) and primary intestinal lymphoma (PIL) are similar, but they have their own characteristics. Therefore, the differential diagnosis is difficult. The diagnosis of CD is more supported by young, long course of the disease, abdominal pain, oral ulcers, perianal lesions, history of surgery (appendectomy, resection), and multi-site lesions. Short disease duration, abdominal mass and single-site lesion are more common in PIL. Biopsy diagnosis rate of PIL is higher than that of CD. In the case of the diagnosis of PIL after CD treatment, misdiagnosis, a combination of the two and drug induction should be considered.

Background To evaluate the efficacy and safety of Infliximab (IFX) in the management of refractory ulcerative colitis (UC) and UC with extra-intestinal manifestations. Methods The clinical data for 36 cases including refractory UC patients and UC patients with special extra-intestinal manifestations, who all accepted treatment with IFX, were analysed retrospectively. Results Among all the 36 cases, 33 were refractory UC (5 with extra-intestinal manifestations) and another 3 with special extra-intestinal manifestations (2 with perianal abscess and 1 with ankylosing spondylitis). After treatment with IFX, 13 patients achieved clinical remission, of whom 3 achieved mucosal healing, 15 achieved clinical response and 8 failed to respond. The total effective rate of IFX treatment was 77.78%, remission rate 36.11%; Among 8 cases with extra-intestinal manifestations, 5 patients‘ extra-intestinal manifestations improved and the efficacy rate was 62.5%. No serious adverse events occurred during observation. Conclusions IFX is effective and safe in the management of refractory UC and extra-intestinal manifestations.

Objectives This study aims to explore the clinical profiles and onset age-related difference of executive dysfunctions and behavioral changes in early drug-naive patients with Parkinson's disease (PD). Methods A cross-sectional analysis on 419 early stage drug-naive PD patients was conducted. The frontal assessment battery (FAB) was used to assess executive functions and the frontal behavioral inventory (FBI) was used to assess behavioral changes. Results Executive dysfunctions were detected in 113 patients (27.0%), and 219 patients (52.3%) displayed varied degrees of behavioral changes. The most frequent affected domain for the FAB was lexical fluency (31.7%), while the three most frequent affected domains for the FBI were apathy (26.0%), irritability (24.3%) and inattention (20.8%). Compared to the early-onset PD (EOPD) patients, the late-onset PD (LOPD) patients exhibited significantly higher frequent damage in FAB especially similarities, motor series, and conflicting instructions as well as lower frequent damage in lexical fluency. The frequencies of FBI-abnormal were not different between the two groups. Multivariate analyses indicated that age at PD onset was independently associated with FAB total score and its subscores including lexical fluency, motor series, conflicting instructions and go-no go task, but it has no relationship with FBI total score and its subscores. Conclusions Executive deficits and behavioral changes are common in the early stage of PD. Age at PD onset is independently associated with the performance of executive functions. However, behavioral changes in PD may be not affected by onset age.

Keloid is an overgrowth of scar tissue that develops around a wound. The mechanisms of keloid formation and development still remain unknown, and no effective treatment is available. Searching for active natural resources may develop better prevention and treatment approaches for keloids. Aspidin PB is a natural resource with lower toxicity. We explored its effect on the regulation of TGF-β1-induced expression of type I collagen, CTGF, and α-SMA in keloid fibroblasts (KFs). Western blotting was used to detect the expression levels of type I collagen, CTGF, α-SMA, PI-3K/Akt and Smad-dependent and Smad-independent signaling pathway. The effect of aspidin PB on cell viability in human keloid fibroblasts was measured by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). The percentage of the apoptotic cells was studied by flow cytometry. Based on our results, we revealed that aspidin PB inhibited the production of type I collagen, CTGF, and α-SMA in TGF-β1-induced KFs by blocking PI-3K/Akt signaling pathway. The TGF-β1-mediated phosphorylated levels of Smad2/3 were inhibited by aspidin PB pretreatment. Conclusively, our study suggests that aspidin PB has an inhibitory effect on fibrogenesis in TGF-β1-induced KFs. Our findings imply that aspidin PB has a therapeutic potential to intervene and prevent keloids and other fibrotic diseases.

Background In order to improve therapy for head and neck squamous cell carcinoma (HNSCC), biomarkers associated with local and/or distant tumor relapses and cancer drug resistance are urgently needed. This study identified a potential biomarker, Bcl-2 associated athanogene-1 (BAG-1), that is implicated in HNSCC insensitive to cisplatin and tumor progression. Methods Primary and advanced (relapsed from parental) University of Michigan squamous cell carcinoma cell lines were tested for sensitivity to cisplatin and gene expression profiles were compared between primary (cisplatin sensitive) and the relapsed (cisplatin resistant) cell lines by using Agilent microarrays. Additionally, differentially expressed genes phosphorylated AKT, and BAG-1, and BCL-xL were evaluated for expression using HNSCC tissue arrays. Results Advanced HNSCC cells revealed resistant to cisplatin accompanied by increased expression of BAG-1 protein. siRNA knockdown of BAG-1 expression resulted in significant improvement of HNSCC sensitivity to cisplatin. BAG-1 expression enhanced stability of BCL-xL and conferred cisplatin resistant to the HNSCC cells. In addition, high levels of expression of phosphorylated AKT, BAG-1, and BCL-xL were observed in advanced HNSCC compared to in that of primary HNSCC. Conclusion Increased expression of BAG-1 was associated with cisplatin resistance and tumor progression in HNSCC patients and warrants further validation in larger independent studies. Over expression of BAG-1 may be a biomarker for cisplatin resistance in patients with primary or recurrent HNSCCs and targeting BAG-1 could be helpful in overcoming cisplatin resistance. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1289-2) contains supplementary material, which is available to authorized users.

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.

Multiple myeloma (MM) cells secrete osteoclastogenic factors that promote osteolytic lesions; however, the identity of these factors is largely unknown. Here, we performed a screen of human myeloma cells to identify pro-osteoclastogenic agents that could potentially serve as therapeutic targets for ameliorating MM-associated bone disease. We found that myeloma cells express high levels of the matrix metalloproteinase MMP-13 and determined that MMP-13 directly enhances osteoclast multinucleation and bone-resorptive activity by triggering upregulation of the cell fusogen DC-STAMP. Moreover, this effect was independent of the proteolytic activity of the enzyme. Further, in mouse xenograft models, silencing MMP-13 expression in myeloma cells inhibited the development of osteolytic lesions. In patient cohorts, MMP-13 expression was localized to BM-associated myeloma cells, while elevated MMP-13 serum levels were able to correctly predict the presence of active bone disease. Together, these data demonstrate that MMP-13 is critical for the development of osteolytic lesions in MM and that targeting the MMP-13 protein - rather than its catalytic activity - constitutes a potential approach to mitigating bone disease in affected patients.

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.

The purpose of the study is to evaluate a new immunochemical fecal occult blood test method (Hemosure IFOBT), and compare it to the Guaiac-based chemical method (CFOBT) for colorectal cancer detection. A hypothetical sequential method (SFOBT), in which IFOBT was used only as a confirmatory test for CFOBT, was also evaluated. A total of 324 patients were recruited from 5 major hospitals in Beijing, China. For each patient, 3 consecutive stool samples were collected for simultaneous CFOBT and IFOBT tests, followed by colonoscopic examination. We compared the sensitivity and specificity of the 3 methods (CFOBT, IFOBT and SFOBT) in two settings, with the first 2 consecutive samples versus all 3 samples. Although the sensitivity for the detection of cancer and large (20 mm) or multiple adenoma was similar for all 3 methods in the three-sample setting, in the two-sample setting IFOBT had higher sensitivity than SFOBT for detecting cancer (87.8% vs. 75.5%, respectively, p0.05) and large (20 mm) or multiple adenomas (65.4% vs. 42.3%, respectively, p0.05). The IFOBT also had a higher specificity than the CFOBT (89.2% vs. 75.5%, respectively, p0.01) in "normal" individuals defined by colonoscopy in the three-sample setting. Comparing two-sample setting to the three-sample setting, both CFOBT and SFOBT showed significant loss of sensitivity for the detection of cancer as well as adenoma, whereas the sensitivity for IFOBT did not change significantly. Overall, IFOBT with two-sample testing showed compatible sensitivity and specificity to the three-sample testing, and had a lower relative cost per cancer detected than the three-sample testing. In conclusion, the new Hemosure IFOBT with two consecutive stool samples appears to be the most cost-effective approach for colon cancer screening.

Introduction A congenital lower lip nevus is common, but a lower lip length lesion of more than 40% is hard to excise simply, without any distortion. We designed a lower lip bilateral advanced skin flap to restore the wound surface after completely and successfully removing the nevus. Case presentation A 13-year-old Mongolian girl was referred to us with a giant congenital lower lip nevus. The lesion covered nearly half of her lower lip. We designed a lower lip bilateral advanced skin flap to restore the wound surface, after completely removing the nevus without advanced repair. Conclusions This case report demonstrates a case of a lower lip length defect of more than 40%, repaired by direct closure without dysfunction. It was a successful attempt that minimized the subsequent scar and dysfunction.

OBJECTIVE: To assess the value of Fourier-transform infrared (FT-IR) spectroscopic analysis in detecting colorectal carcinoma. METHODS: Fifty pairs of specimens of exfoliated cells from patients with colorectal carcinoma, who had undergone colorectal resection, were analyzed by using FT-IR spectrometry. Exfoliated cells were obtained by scraping off: (i) the foci of the colorectal carcinoma; and (ii) the normal mucosa more than 5 cm away from the foci. Cell specimens were fixed and smeared onto two slides: a gold-coated slide for the infrared spectrometry and a conventional one for histopathological evaluation. The spectra from 28 pairs of specimens were analyzed by logistic regression with multiple factors to establish a statistical model for predicting the probability of colorectal cancers, and then the spectra of the remaining 22 pairs were judged according to the model. Their corresponding diagnostic accuracy was calculated. RESULTS: Substantial differences were found in the spectral properties of cancer and non-cancer cells, which were evident in the frequency region of 1000–1350/cm. A shift of the phosphodiester groups of nucleic acids was noted: compared with the spectral features of non-cancerous cells, the distance between VsPO2– and VasPO2– was shorter in cancerous cells. The C–O stretching bonds of cell proteins were significantly changed in the frequency region of 1140–1180/cm. In non-cancerous cells, the peak intensity of 1155/cm was stronger than that of 1174/cm, whereas in cancerous cells the peak intensity of 1155/cm was weaker than that of 1174/cm; the peak intensity ratio of 1174/cm : 1155/cm was increased in cancerous cells. The spectral characteristics of the spectrometric data from the 22 pairs of specimens were consistent with those of the base-model data. The other 22 pairs of specimens were analyzed according to the model, indicating that Fourier-transform infrared spectrometry can be used to discriminate cancerous cells from non-cancerous cells with a sensitivity of 90.91%, a specificity of 86.36%, a positive prediction value of 86.96% and a negative predictive value of 90.48%. CONCLUSIONS: Fourier-transform infrared spectrometry is of value in detecting colorectal cancer cells and it could be used as a rapid method for the clinical screening of malignant cells.

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: c-Myc is one of the most frequently altered genes across a vast array of human cancers. Overexpression of c-Myc is observed in up to 30% of cases of diffuse large B-cell lymphoma (DLBCL), the most common type of aggressive lymphoma. Although DLBCL can be cured in 60-70% patients, a substantial minority of patients with DLBCL still die from their lymphoma. There is emerging evidence that c-Myc expression is an adverse risk factor independent of the cell-of-origin classification. To date no drugs that directly target c-Myc have been approved for the treatment of any cancer. In fact, since c-Myc is involved in many normal cellular functions, direct c-Myc inhibitors may be associated with significant toxicity. The goal of our study is to develop novel strategies targeting c-Myc that will have an improved therapeutic index. The c-Myc protein has a very short half-life of less than 30 minutes, and its translation is highly dependent on the eukaryotic initiation factor 4F (eIF4F). eIF4F is activated by the mammalian target of rapamycin (mTOR), which is regulated not only by the PI3K-AKT pathway but also the proteasome pathway. These observations led us to hypothesize that if the proteasome and PI3K pathways cooperate in the activation of mTOR and its downstream target eIF4F, then combinations of proteasome and PI3K inhibitors should potently suppress eIF4F dependent translation of c-Myc and the growth of c-Myc dependent lymphoma. Methods: Cytotoxicity was studied in lymphoma cell lines and primary lymphoma cells using Cell TiterGlo. The Bliss additivism model was used to determine the expected inhibition of cell growth. The difference of the expected and observed levels of inhibition was used to calculate the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Mechanisms of synergy were determined through interrogation of PI3K/AKT/mTOR pathway and its downstream targets. 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. Results: We used a high-throughput platform to screen the cytotoxicity of two PI3Kdelta inhibitors (TGR-1202 & idelalisib/Cal-101), two proteasome inhibitors (bortezomib & carfilzomib), and four combination pairs using these drugs. We found that TGR-1202 and Cal-101 caused only minimal to mild inhibition of lymphoma cells, while bortezomib and carfilzomib caused potent inhibition as single agents. The combination of TGR-1202 and carfilzomib was consistently the most synergistic doublet, while the combination of Cal-101 and bortezomib the least synergistic in numerous lymphoma cell lines studied to date (Figure 1). TGR-1202 and carfilzomib were also highly synergistic in primary lymphoma cells, while Cal-101 and bortezomib were not. Importantly, normal lymphocytes were resistant to the combination of TGR-1202 and carfilzomib. At the molecular level, only the combination of TGR-1202 and carfilzomib potently inhibited mTORC1 dependent phosphorylation of 4E-BP1, leading to marked reduction of c-Myc protein (Figures 2 & 3). In contrast, the combination of TGR-1202 and carfilzomib produced no reduction of the mRNA level of c-Myc (Figures 3). A luciferase reporter demonstrated that the synergistic combination TGR-1202 and carfilzomib specifically inhibited the translation downstream of the 5'UTR of c-Myc (Figure 3). GEP studies confirmed that the canonical c-Myc target genes were potently downregulated at the level of transcription by the combination of TGR-1202 and carfilzomib (Figure 3). These results demonstrate that TGR-1202 and carfilzomib in combination potently inhibited the translation of c-Myc and the c-Myc transcriptional program, which appears to be primarily through disruption of the PI3Kdelta and proteasome pathways that converge on mTOR. Ongoing experiments are focused on confirmation of these observations in vivo. Further, a phase I/II clinical trial evaluating this combination regimen in aggressive c-Myc driven lymphomas is being planned. Disclosures Deng: TG Therapeutics: Research Funding; Gilead: Research Funding; Amgen/Onyx: Research Funding. Lentzsch:Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Axiom: Honoraria. O'Connor:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Research Funding; Acetylon: Consultancy, Other: Consultancy fee; Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Other: Consultancy fee; Takeda Millenium: Consultancy, Honoraria, Other: Consultancy fee, Research Funding; Novartis: Consultancy, Honoraria, Other: Consultancy fee; Seattle Genetics: Research Funding; Bayer: Consultancy, Honoraria.

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.

Immunomodulatory derivatives of thalidomide (IMiDs) have been used for the treatment of myelodysplastic syndrome and multiple myeloma; however, the mechanism of action of IMiDs is largely unknown. The purpose of this review is to provide an overview of recent findings on the mechanism of action of IMiDs, its use as a new treatment modality for various hematologic malignancies, and problems associated with stem cell mobilization after lenalidomide treatment.Recent clinical trials revealed lenalidomide as a promising new agent for the treatment of follicular non-Hodgkin's lymphoma (NHL) and also diffuse large B-cell lymphoma. Pomalidomide was shown to be even more effective in refractory multiple myeloma than lenalidomide. New guidelines for the management of venous thromboembolism have been established. The chemokine receptor 4 (CXCR4) inhibitor, AMD-3100, is recommended for patients who have received lenalidomide and failed to mobilize stem cells after G-SCF and cyclophosphamide. Preclinical studies investigated the pleiotropic functions of IMiDs, with a particular focus on immune modulation, their effects on new targets, stem cells and disruption of plasma cell microenvironment interactions.More and more indications for the use of IMiDs in hematologic malignancies have been identified. In order to establish better clinical usage of IMiDs, it is of utmost importance to clarify the antitumor mechanism of IMiDs. Here, we provide a review on the recent advances in the development of IMiDs. New guidelines for venous thromboembolism prophylaxis and stem cell mobilization failure associated with lenalidomide treatment have been established.

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.

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.

Colorectal cancer is one of the most prevalent malignancies in the world. Chinese researchers and clinical doctors had been working hard in the last 12 years, exploring efficient and convenient methods for screening and early diagnosis. The 12 years research works indicated that SFOBT plus colonoscopy was the best detecting protocol for mass screening of colorectal cancer. Serial combined testing for tumor markers plus colonoscopy is the most commonly used strategy for early diagnosis. However, still no optimum method for early diagnosis was generally accepted by most researchers. Due to intrinsic defects of the techniques, several screening and diagnostic methods should be combined to bring each advantage into full play, in order to elevate the diagnostic level of colorectal cancer.

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.

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.

8099 Background: MM cells produce OCL-activating factors that induce excessive bone resorption resulting in lytic lesions. The role of MMPs in invasion/progression of solid tumors is well-known, but its function in MM has not been well elucidated. Our group has shown that MMP13 is highly expressed in primary MM cells and in sera of MM patients. Levels of MMP13 significantly correlate with the extent of bone disease. MMP13 is induced by IL-6 via AP-1 activation in MM cells and enhances fusion of OCL precursors resulting in excessive bone resorption. OCL formation using MNCs of mmp-13-/- mice resulted in a fusion defect, significantly decreased OCL size and activity, which could be reversed by exogenous MMP13 (ASH 2009, IMW 2011). Methods: Methods will be presented in the Results section. Results: RT-PCR and western blotting revealed that IL-6 treatment of MM cells induced MMP13 transcription (30-fold) and secretion (>1000-fold). Protein expression of the AP-1 members c-Jun and c-Fos was induced by IL-6, which correlated with MMP13 upregulation. Our data further indicate that the catalytic activity of MMP13 is not required to enhance OCL formation and bone resorption. To prove this, we generated the MMP13 activity-dead mutation MMP13-E223A construct by site-directed mutagenesis PCR-based cloning. The mutated protein was overexpressed in HEK293 cells and purified from the supernatant to confirm whether loss of catalytic activity blocks MMP13 function. To further investigate the in vivo role of MMP13 in MM bone disease, MMP13 expression was knocked down (KD) in murine 5TGM1-MM cells by pKLO. 1 puro lentiviral infection containing sh-RNA targeting mouse MMP13 sequence. MMP13-KD 5TGM1-MM cells or WT-5TGM1-MM cells were intratibially injected into RAG2-/- mice. Development of lytic bone lesions are monitored by micro-QCT and data will be available at the time of presentation. Conclusions: Our data suggest that MMP13, secreted by MM cells, plays a critical role in the development of lytic lesions. Targeting MMP13 represents a promising approach to treat or to prevent bone disease in MM.

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 1840 Introduction: The IMiD® immunomodulatory drugs lenalidomide (LEN) and pomalidomide (POM) yield high response rates in patients with multiple myeloma (MM). However, the use of IMiDs is associated with neutropenia and thrombocytopenia. It has been shown before that IMiDs down-regulate the transcription factor PU.1/SPI1 leading to maturational arrest of granulocytes with accumulation of immature myeloid precursors and subsequent neutropenia (Blood. 2010; 115:605-614). However, the mechanism underlying the development of thrombocytopenia is unclear. Methods and Results: Here, we investigated the effects of IMiDs on megakaryopoiesis. In megakaryocytic colony formation assays IMiDs significantly (p The finding that IMiDs induce development of megakaryocytic precursors is in contrast to the clinical observation that IMiDs induce thrombocytopenia. Since previous studies showed that IMiDs are not directly toxic to bone marrow hematopoietic cells (Blood. 2005; 105:3833-40), we hypothesized that these effects might be induced by a maturational arrest of megakaryopoiesis resulting in thrombocytopenia. Therefore we studied the morphology of Mks for signs of immaturity. Giemsa staining and CD61 immunohistochemistry showed Mks developed in the presence of IMiDs were smaller in size with hypo-lobulated nuclei. 45% of cells treated with LEN, and 52% treated with POM exhibited a single nuclear lobe compared to 23% of cells treated with DMSO. We confirmed our findings by measuring the ploidy of cultured Mks by FISH. Mks (CD61-FITC-positive) were studied from each treatment (DMSO and IMiDs) and the number of nuclei and the number of centromeric region of chromosome 6 (CEP 6) signals for each Mk were recorded. CD61-FITC-negative cells were omitted from the FISH analyses. The percentage of 2N (disomic) cells was 2.5-fold higher (p=0.0221) in the IMiDs group compared to control group; in contrast the percentage of 4N and ≥8N cells (with multiple disomic nuclei) decreased 1.5 and 1.3-fold (p Since failure of terminal differentiation and excessive proliferation of Mks has been described in GATA1-deficient Mks (Blood. 2005; 106:1223-1231) we analyzed the expression of GATA1 at the mRNA and protein levels after treatment with IMiDs using real-time PCR, Western blot and immunofluorescence microscopy, respectively. IMiDs-treated Mks showed down-regulation of GATA1 associated with decreased expression of FOG1/ZFPM1 and NFE2 both critically interacting with GATA1. Further, previous studies suggested that polyploidy formation in Mks depends on the expression of cyclin D1 isotypes (Blood. 2007; 109:5199-5207). Indeed, IMiDs treatment caused a decrease of p21 and CCND1. Conclusion: Taken together, our findings suggest that IMiDs maintain and expand early hematopoietic progenitors up to several weeks. Our data further indicate that the loss of key transcription factors such as GATA1 and CCND1 both critical for megakaryopoiesis precludes Mks from continued maturation. This might lead to maturational arrest of Mks with accumulation of immature megakaryocytic precursors and subsequent thrombocytopenia. Disclosures: Lentzsch: Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.

Abstract 135 Background: Lenalidomide and pomalidomide are IMiD® immunomodulatory compounds that have been shown to be highly active in the treatment of multiple myeloma (MM). IMiD compounds exert their anti-tumor effects via acting on costimulatory proteins of T cells and NK cells, augmenting both the adaptive and innate immune system. But the mechanisms by which IMiD compounds directly inhibit MM cell proliferation are still unclear. Here we focused on the direct effects of IMiD compounds alone on MM cells. Results and Methods: We found that IMiDs, at concentrations as low as 0.01 μ M, induce significant inhibition of DNA synthesis in MM cells as shown by thymidine uptake. Since our previous work demonstrated that C/EBPβ is an important transcription factor which controls the growth and proliferation of myeloma cells, we analyzed the effects of IMiD compounds on C/EBPβ. We found that both pomalidomide and lenalidomide significantly decreased the protein level of C/EBPβ LAP-isoform in MM cell lines and primary MM cells. IMiD compound-induced suppression of C/EBPβ protein expression led to impaired transcription of the downstream IRF4, and subsequently to downregulation of BLIMP1 and XBP1, which are all critical for MM survival. To confirm our findings in vivo, we analyzed IRF4 expression by double labeling (IRF4+/CD138+) immunohistochemical staining of bone marrow biopsy samples of 23 myeloma patients prior to therapy and during therapy with lenalidomide. During lenalidomide therapy, the bone marrow MM cells showed a significantly weaker staining intensity for IRF4 in comparison to prior therapy. This was quantified by a significant (p Conclusions: Our studies, for the first time, provide evidence that IMiD compounds inhibit MM cell proliferation and survival by affecting the translation of C/EBPβ and subsequently multiple downstream transcription factors including IRF4, BLIMP1 and XBP1. Due to the critical role of C/EBPβ in mediating effects of IMiD compounds in MM, it might be a target to overcome drug resistance to IMiD compounds. The fact that pomalidomide can overcome resistance to lenalidomide in MM requires still further evaluation. Disclosures: Schafer: Celgene Corporation: Employment, Equity Ownership. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.

Abstract 4079 Background: In multiple myeloma (MM), lenalidomide has impressive clinical activity in patients with both relapsed/refractory and newly diagnosed disease. Nevertheless hematopoietic stem cell transplantation (HSCT) is still the “backbone” in the treatment of newly diagnosed MM patients and very often lenalidomide treatment (as induction therapy) is combined with HSCT in order to achieve high response rates. Clinical data have shown that in up to 43% of those patients, standard mobilization of CD34+ cells with G-CSF alone failed to mobilize significant numbers of hematopoietic progenitors into peripheral blood, raising concerns about potential stem cell toxicity of lenalidomide (Mazumder et al, Leukemia 2008). Interestingly mobilization of hematopoietic progenitors with AMD-3100 (Plerixafor) overcomes mobilization failures in almost all patients previously treated with lenalidomide. The fact that AMD-3100 antagonizes the binding of chemokine stromal- cell–derived factor-1α (SDF-1α) to CXC chemokine receptor 4 (CXCR4) suggests a potential role of the CXCR4/SDF-1α axis in mediating mobilization failure after lenalidomide treatment. Subsequently, the findings noted above raised questions on: 1) the stem cell toxicity of lenalidomide; 2) the underlying mechanism that mediates the development of G-CSF resistance; and 3) the mechanism of the modulation of the CXCR4/SDF-1α axis by lenalidomide. In our previous work we showed the following. 1) Lenalidomide neither inhibited colony formation in standard colony assays nor the development of cobble stone area forming cells (CAFC) in LTC-IC assays, suggesting that lenalidomide is not stem cell toxic (Koh et al, Blood 2005). 2) We further showed that lenalidomide significantly upregulated G-CSF secretion of CD34+ cells (600%), suggesting that the high levels of G-CSF may mediate a relative resistance towards G-CSF-induced mobilization (Pal et al, Blood, 2010). Results and Methods: We analyzed why blocking CXCR4 by AMD-3100 overcomes mobilization failure to G-CSF. We first examined the CXCR4 expression profile of CD34+ cells treated with lenalidomide. Lenalidomide treatment significantly upregulated the expression of CXCR4 on cell surface after 48h treatment measured by flow cytometry. Increased expression of CXCR4 onCD34+ cells remained high with continuous treatment. Western blot assay of the hydrophobic (membrane) and the hydrophilic (cytosol) cell fraction confirmed our data showing that lenalidomide increases the expression of CXCR4 on CD34+ cell surface. Confocal microscopy showed that lenalidomide inhibited SDF-1α induced CXCR4 internalization. In accordance with the increased CXCR4 surface expression, transwell migration assay revealed that the SDF-1α-induced migration of CD34+ cells in the presence of lenalidomide significantly increased by 52% in comparison to control. Quantitation of SDF-1α showed that CXCR4 had no significant effect on the secretion of SDF-1α by CD34+ cells and in human stromal cells. Result: In conclusion, our data show that lenalidomide is not toxic to hematopoietic progenitors. The strong increase of G-CSF secretion by CD34+ cells might contribute to a desensitization of CD34+ cells to G-CSF mobilization. Primarily our data indicate that increased expression of CXCR4 followed by blocked internalization, increases binding to SDF-1α secreted by the bone marrow niche. This subsequently prohibits the mobilization of CD34+ cells. These data suggest that blocking the CXCR4 receptor by AMD-3100 disrupts this circle and finally permits the mobilization of hematopoietic cells from the bone marrow niche into peripheral blood. This study provides novel insights into the effects of lenalidomide on CD34+ cells relevant for HSCT in MM. Disclosures: Roodman: Amgen: Consultancy; Celgene Corp: Consultancy; Acceleron: Consultancy; Millennium: Consultancy. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.

Abstract 1844 Poster Board I-870 Background: Lenalidomide is an immunomodulatory drug that has been shown to be highly active in the treatment of multiple myeloma (MM). In combination with dexamethasone, remission rates of up to 61% are achieved in newly diagnosed MM patients. Nevertheless, the mechanisms of how immunomodulatory compounds induce high remission rates in MM remains elusive. Methods and Results: Here we investigate the underlying anti-MM mechanism of lenalidomide. Lenalidomide significantly (p Disclosures: Schafer: Celgene Corporation: Employment. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.