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Background aims The vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) signaling pathway plays an important role in angiogenesis and lymphangiogenesis, which are closely related to tumor cell growth, survival, tissue infiltration and metastasis. Blocking/interfering with the interaction between VEGF and VEGFR to inhibit angiogenesis/lymphangiogenesis has become an important means of tumor therapy. Methods Here the authors designed a novel chimeric antigen receptor (CAR) lentiviral vector expressing the VEGF-C domain targeting both VEGFR-2 and VEGFR-3 (VEGFR-2/3 CAR) and then transduced CD3-positive T cells with VEGFR-2/3 CAR lentivirus. Results After co-culturing with target cells, VEGFR-2/3 CAR T cells showed potent cytotoxicity against both VEGFR-2- and VEGFR-3-positive breast cancer cells, with increased simultaneous secretion of interferon gamma, tumor necrosis factor alpha and interleukin-2 cytokines. Moreover, CAR T cells were able to destroy the tubular structures formed by human umbilical vein endothelial cells and significantly inhibit the growth, infiltration and metastasis of orthotopic mammary xenograft tumors in a female BALB/c nude mice model. Conclusions The authors’ results indicate that VEGFR-2/3 CAR T cells targeting both VEGFR-2 and VEGFR-3 have significant anti-tumor activity, which expands the application of conventional CAR T-cell therapy.

Regulatory T cells (Tregs) could maintain the characteristics of stem cells and inhibit the differentiation of normal hematopoietic stem/progenitor cells. Recent studies have shown that Tregs, as an important component of acute myeloid leukemia (AML) microenvironments, can help AML cells to evade immune surveillance. However, their function in directly regulating the stemness of AML cells remains elusive. In this study, the increased stemness of AML cells promoted by Tregs was verified in vitro and in vivo. The cytokines released by Tregs were explored, the highly expressed anti-inflammatory cytokine IL10 was found, which could promote the stemness of AML cells through the activation of PI3K/AKT signal pathway. Moreover, disrupting the IL10/IL10R/PI3K/AKT signal in AML/ETO c-kitmut (A/Ec) leukemia mice could prolong the mice survival and reduce the stemness of A/Ec leukemia cells. Finally, it was confirmed in patient samples that the proportion of Tregs to leukemia stem cells (LSCs) was positively correlated, and in CD34+ primary AML cells, the activation of PI3K/AKT was stronger in patients with high Tregs’ infiltration. After rhIL10 treatment, primary AML cells showed increased activation of PI3K/AKT signaling. Therefore, blocking the interaction between Tregs and AML cells may be a new approach to target LSCs in AML treatment.

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation involving RARα and its fusion partners. For decades, the advent of all-trans retinoic acid (ATRA) synergized with arsenic trioxide (As2O3) has turned most APL from highly fatal to highly curable. TBLR1-RARα (TR) is the tenth fusion gene of APL identified in our previous study, with its oncogenic role in the pathogenesis of APL not wholly unraveled. In this study, we found the expression of TR in mouse hematopoietic progenitors induces blockade of differentiation with enhanced proliferative capacity in vitro. A novel murine transplantable leukemia model was then established by expressing TR fusion gene in lineage-negative bone marrow mononuclear cells. Characteristics of primary TR mice revealed a rapid onset of aggressive leukemia with bleeding diathesis, which recapitulates human APL more accurately than other models. Despite the in vitro sensitivity to ATRA-induced cell differentiation, neither ATRA monotherapy nor combination with As2O3 confers survival benefit to TR mice, consistent with poor clinical outcome of APL patients with TR fusion gene. Based on histone deacetylation phenotypes implied by bioinformatic analysis, HDAC inhibitors demonstrated significant survival superiority in the survival of TR mice, yielding insights into clinical efficacy against rare types of APL.

Pharmacological stimulation of adipose tissue remodeling and thermogenesis to increase energy expenditure is expected to be a viable therapeutic strategy for obesity. Berberine has been reported to have pharmacological activity in adipose tissue to anti-obesity, while the mechanism remains unclear. Here, we observed that berberine significantly reduced the body weight and insulin resistance of high-fat diet mice by promoting the distribution of brown adipose tissue and thermogenesis. We have further demonstrated that berberine activated energy metabolic sensing pathway AMPK/SIRT1 axis to increase the level of PPARγ deacetylation, which leads to promoting adipose tissue remodeling and increasing the expression of the thermogenic protein UCP-1. These findings suggest that berberine that enhances the AMPK/SIRT1 pathway can act as a selective PPARγ activator to promote adipose tissue remodeling and thermogenesis. This study proposes a new mechanism for the regulation of berberine in adipose tissue and offers a great prospect for berberine in obesity treatment.

Background aims Anti-CD19 chimeric antigen receptor (CAR)-modified T cells have shown dramatic cytotoxicity against B-cell malignancies. Currently, autologous T cells are conventionally used to manufacture CAR T cells. Low quality or insufficient quantity of autologous T cells may lead to failure of CAR T preparations. Moreover, CAR T preparation usually takes 1–2 weeks, which is too long for patients with rapid disease progression to successfully infuse CAR T cells. Thus, the development of a ready-to-use CAR immunotherapy strategy is needed. NK-92, a natural killer (NK) cell line derived from an NK lymphoma patient, has been gradually applied as a CAR-modified effector cell. To avoid the potential development of secondary NK lymphoma in patients, large doses of radiation are used to treat NK-92 cells before clinical application, which ensures the safety but reduces the cytotoxicity of NK-92 cells. Therefore, it is crucial to explore a suitable radiation dose that ensures short life span and good cytotoxicity of CAR NK-92 cells. Methods NK-92MI, a modified IL-2-independent NK-92 cell line, was used to establish an anti-CD19 CAR NK. The suitable radiation dose of CAR NK was then explored in vitro and validated in vivo, and the specific cytotoxicity of irradiated and unirradiated CAR NK against CD19+ malignant cells was assessed. Results CAR NK exhibited specific cytotoxicity against CD19+ malignant cells. Irradiation ensured a short life span of CAR NK in vitro and in vivo. Encouragingly, irradiated CAR NK displayed an anti-CD19+ malignancy capacity similar to that of unirradiated CAR NK. Conclusions Five Gy is a suitable radiation dose to ensure the safety and effectiveness of CD19 CAR NK-92MI cells.

Acute myeloid leukemia (AML) is a biologically and clinically heterogeneous disease with a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B cell malignancies but a dismal consequences in AML. In our previous study, we found that interleukin-10 receptor (IL-10R) was overexpressed in most AML cells, and played an important role in promoting the stemness of leukemia cells. In this study, we developed a novel ligand-based CAR-T cell targeting IL-10R, which displayed striking cytotoxicity both in vitro and in vivo against AML cells. Except for monocytes, it had no significant adverse effects on the normal hematopoietic system, including CD34+hematopoietic stem and progenitor cells (HSPCs). In addition, even though the incorporation of IL-10 in the CAR cassette led to phenotypes change, it had few adverse effects on the survival and biological activity of IL-10 CAR-T cells and did not cause excessive proliferation of leukemia cells. Therefore, we propose IL-10R is a novel promising therapeutic candidate for AML, and IL-10R targeted CAR-T therapy provides a new treatment strategy to improve the prognosis of AML.

Background: Although increasing data suggest that hyperthyroidism is associated with adverse pregnancy outcomes, there are only a few reports with different conclusions on whether the mild...

Aim: Although numerous pro-inflammatory cytokines promote signaling via intracellular pathways involving Janus kinases, it remains unclear if ruxolitinib, a Janus kinase1/2 inhibitor, provides control of cytokine-release syndrome (CRS) without toxicity against therapeutic T cells. Materials & methods: We report successful clinical experience using ruxolitinib as adjuvant therapy to treat steroid-refractory CRS, which was related to CD22/CD19 chimeric antigen receptor-modified T cell sequential infusion, in a patient with Philadelphia chromosome-like acute lymphoblastic leukemia. Results: His symptoms improved rapidly after first dose of ruxolitinib; this was associated with reduced levels of circulating pro-inflammatory indicators. He eventually achieved minimal residual disease negative remission. Discussion: This is the first case in which ruxolitinib was used to treat steroid-refractory CRS; furthermore, this intervention had no apparent impact on the antileukemic actions of the chimeric antigen receptor-modified T cells. Our results suggest that adjuvant ruxolitinib therapy may be an alternative therapeutic approach for the management of CRS.

// Haiying Niu 1, * , Wei Zhou 2, * , Yingxi Xu 3 , Zhiqi Yin 4 , Wenzhi Shen 2 , Zhen Ye 2 , Yanhua Liu 2 , Yanan Chen 2 , Shuang Yang 2 , Rong Xiang 2 , Lina Wang 3 and Pengpeng Qu 5 1 Department of Gynecology and Obstetrics, Tianjin First Center Hospital, Tianjin, China 2 Department of Immunology, Nankai University School of Medicine, Tianjin, China 3 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences, Tianjin, China 4 Department of Pathology, Tianjin First Center Hospital, Tianjin, China 5 Department of Gynecology Oncology, Tianjin Central Hospital of Gynecology Obstetrics, Tianjin, China * These authors have contributed equally to this work Correspondence to: Lina Wang, email: wanglina@ihcams.ac.cn Pengpeng Qu, email: qu.pengpeng@hotmail.com Keywords: PPA1, epithelial ovarian cancer, metastasis, Wnt/β-catenin Received: January 18, 2017 Accepted: June 19, 2017 Published: July 18, 2017 ABSTRACT Inorganic pyrophosphatase (PPA1) activity is a key determinant of cellular inorganic pyrophosphate levels, and its expression is correlated with growth of several solid tumors. To investigate this relationship, we first examined PPA1 expression in human epithelial ovarian cancer (EOC) samples, and found that PPA1 was overexpressed in tumors from EOC patients. Higher PPA1 levels correlated with advanced grades, stages, and poor survival in EOC patients. Examination of PPA1 function in EOC revealed that silencing PPA1 inhibited EOC migration, epithelial-mesenchymal transition (EMT), and metastasis in vitro and in vivo . In addition, PPA1 may promote the dephosphorylation and translocation of β-catenin. These results demonstrate that silencing PPA1 inhibits EOC metastasis by suppressing the Wnt/β-catenin signaling pathway. Strategies for downregulating PPA1 may have therapeutic potential for the prevention and treatment of EOC.

// Shuang Liu 1 , Wenting Lu 1 , Shouyun Li 1 , Saisai Li 1 , Jia Liu 1 , Yuanyuan Xing 2 , Shuzu Zhang 2 , Joe Zhongxiang Zhou 2 , Haiyan Xing 1 , Yingxi Xu 1 , Qing Rao 1 , Chengjun Deng 2 , Min Wang 1 , Jianxiang Wang 1 1 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China 2 Fujian Jinler Pharmaceuticals, Jiangle County, Fujian 353300, China Correspondence to: Chengjun Deng, email: cjdeng@yahoo.com Min Wang, email: wangjxm@ihcams.ac.cn Jianxiang Wang, email: wangjx@ihcams.ac.cn Keywords: LSD1 inhibitor, leukemia, proliferation inhibition, apoptosis, autophagy Received: December 09, 2016 Accepted: March 15, 2017 Published: March 29, 2017 ABSTRACT Lysine-specific demethylase 1 (LSD1) has been recognized as a potential therapeutic target for acute myeloid leukemia (AML). Herein, we identified a novel LSD1 inhibitor, JL1037, via Computer Aided Drug Design technology. JL1037 is a potent, selective and reversible LSD1 inhibitor with IC50s of 0.1 μM and >1.5 μM for LSD1 and monoamine oxidases A/B (MAO-A/B), respectively. Treatment of THP-1 and Kasumi-1 cell lines with JL1037 resulted in dose dependent accumulation of H3K4me1 and H3K4me2, the major substrates of LSD1, as well as inhibition of cell proliferation, blockade of cell cycle and induction of apoptosis. Further investigations demonstrated that JL1037 could upregulate cell cycle-related proteins P21, P57, pro-apoptotic protein Bax and downregulate anti-apoptosis proteins Bcl-2 and Bcl-XL. JL1037 appeared to activate autophage response in AML cell lines as well as primary cells from AML patients by increasing LC3-II expression and the formation of autophagosomes and autolysosomes in cytoplasm. Co-treatment with autophagy inhibitor chloroquine (CQ) enhanced JL1037-induced cell apoptosis. Moreover, daily intravenous administration of JL1037 tended to reduce tumor burden and prolong the survival of t(8;21) leukemia mice. In conclusion, JL1037 exhibited potent anti-leukemia effect and could be a potential therapeutic agent for AML treatment.

// Wenting Lu 1 , Tengteng Yu 1 , Shuang Liu 1 , Saisai Li 1 , Shouyun Li 1 , Jia Liu 1 , Yingxi Xu 1 , Haiyan Xing 1 , Zheng Tian 1 , Kejing Tang 1 , Qing Rao 1 , Jianxiang Wang 1 and Min Wang 1 1 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China Correspondence to: Jianxiang Wang, email: wangjx@ihcams.ac.cn Min Wang, email: wangjxm@ihcams.ac.cn Keywords: FHL2, iASPP, biological functions, leukemia Received: December 23, 2016 Accepted: March 09, 2017 Published: March 28, 2017 ABSTRACT iASPP is an inhibitory member of apoptosis-stimulating proteins of p53 (ASPP) family, which inhibits p53-dependent apoptosis. iASPP was highly expressed in acute leukemia, inhibited leukemia cells apoptosis and promoted leukemogenesis. In order to clarify its mechanism, a yeast two-hybrid screen was performed and FHL2 was identified for the first time as one of the binding proteins of iASPP. FHL2 was highly expressed in K562 and Kasumi-1 cells. FHL2 and iASPP interacted with each other and co-localized in both nucleus and cytoplasm. Either FHL2 or iASPP silenced could reduce cell proliferation, induce cell cycle arrest at G0/G1 phase, and increase cell apoptosis. Western blot analysis showed that the level of p21 and p27 increased, CDK4, E2F1, Cyclin E and anti-apoptotic proteins Bcl-2 and Bcl-xL reduced. Interestingly, when FHL2 was knocked down, the protein expression level of iASPP also decreased. Similarly, the expression of FHL2 would reduce when iASPP was silenced. These results indicated that FHL2 might be a novel potential target for acute myelocytic leukemia treatment.

Vitamin D receptor (VDR) plays an important role in regulating energy metabolism. Adipose tissue is a vital metabolic organ in energy balance and glucose homeostasis. In this study, we investigated the role of VDR expressed on adipose tissue in the balance of energy metabolism. This study was conducted in VDR-KO mice, VDR-Tg mice and wild-type mice. Energy metabolism was determined based on the energy expenditure, oxygen consumption, respiratory exchange rate, food and water intake, and a cool room test. VDR expression in the tissues of VDR-Tg mice was assessed by western blotting. The levels of total cholesterol, triglycerides, free fatty acids, leptin, and glucose were assessed using the respective kits. Insulin resistance in the whole body was evaluated by an intraperitoneal glucose tolerance test and insulin tolerance test. mRNA associated with energy metabolism expression in adipose and skeletal muscle tissue was examined by RT-PCR. Our results show that overexpression of VDR in adipose tissue induced an increase in body weight, fat mass, and serum lipid levels, and a decline in energy metabolism; these changes were ameliorated by VDR-KO mice. Overexpression of VDR in the adipose tissue of VDR-Tg mice induced a state of insulin resistance, which corresponded with decreased expression of UCP1, UCP2, UCP3, CPT2 and HK in both white adipose tissue and brown adipose tissue; these changes were also reversed by VDR-KO mice. Our study confirms that expression levels of VDR in adipose tissue play pivotal roles in energy balance and glycolipid metabolism by regulating adipose tissue remodeling.

Cytotoxic chemotherapies could cause the dysregulation of hematopoiesis and even put patients at increased risk of hematopoietic malignancy. Therapy-related leukemia is mainly caused by cytotoxic chemotherapy-induced genetic mutations in hematopoietic stem/progenitor cells (HSPCs). In addition to the intrinsic mechanism, some extrinsic events occurring in the bone marrow (BM) microenvironment are also possible mechanisms involved in genetic alteration. In the present study, we investigated the damage to BM stromal cells induced by a chemotherapy drug, daunorubicin (DNR) and further identified the DNA damage in hematopoietic cells caused by drug-treated stromal cells. It was found that treatment with DNR in mice caused a temporary reduction in cell number in each BM stromal cell subpopulation and the impairment of clonal growth potential in BM stromal cells. DNR treatment led to a tendency of senescence, generation of intracellular reactive oxygen species, production of cytokines and chemokines, and dysfunction of mitochondrial in stromal cells. Transcriptome microarray data and gene ontology (GO) or gene set enrichment analysis (GSEA) showed that differentially expressed genes that were down-regulated in response to DNR treatment were significantly enriched in mitochondrion function, and negative regulators of reactive oxygen species. Surprisingly, it was found that DNR-treated stromal cells secreted high levels of H2O2 into the culture supernatant. Furthermore, coculture of hematopoietic cells with DNR-treated stromal cells led to the accumulation of DNA damage as determined by the levels of histone H2AX phosphorylation and 8-oxo-2′-deoxyguanosine in hematopoietic cells. Overall, our results suggest that DNR-induced BM stromal cell damage can lead to genomic instability in hematopoietic cells.

Background Chimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CAR-T cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CAR-T cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering. Methods Two second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5+ malignant cell lines, primary CD5+ malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5+ T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo. Results Both BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5+ malignant cells in vitro and prolonged the survival of T-ALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5+ malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK. Conclusions Anti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cell-associated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment. Electronic supplementary material The online version of this article (10.1186/s13045-019-0732-7) contains supplementary material, which is available to authorized users.

Rationale: Chimeric antigen receptor-modified T-cell (CART) therapy has revolutionized the treatment of patients with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). However, the capacity of CART therapy has not yet been fully elucidated. Patient concerns: An 18-year-old Chinese male patient presented with multiple firm masses on the skin all over his body following regular chemotherapy. Diagnoses: Bone marrow smear and skin biopsy confirmed that it was a bone marrow and skin relapse from the initial B-cell ALL. Interventions: CD19 CART-cell therapy was performed to manage the bone marrow and skin of the relapsed B-cell ALL. Outcomes: During CART-cell therapy, cytokine release syndrome and central nervous encephalopathy occurred. Eventually, the lesions disappeared, and the bone marrow and skin tested minimal residual disease (MRD) negative. The patient achieved complete remission (CR). Fourteen days after testing MRD negative, he received allogeneic hematopoietic stem-cell transplantation and has remained disease free to date. Lessons: The CR of this patient with leukemia cutis demonstrated that CART exhibited efficacy in this case. While further research is still required, this treatment could potentially be used as a therapy for skin leukemia, lymphoma, and other primary skin cancers.

Background Chimeric antigen receptor-engineered T (CAR-T) cells have extraordinary effect in treating lymphoblastic leukemia. However, treatment of acute myeloid leukemia (AML) using CAR-T cells remains limited to date. Leukemogenesis always relates with the abnormalities of cytogenetics, and nearly one third of AML patients have activating mutations in Fms-like tyrosine kinase 3 (FLT3) which reminded poor prognosis. Considering the FLT3 expressed in AML patients’ blast cells, it may be a new candidate target for CAR-T therapy to treat FLT3+ AML, especially patients harboring FLT3-ITD mutation. Methods The FLT3L CAR-T using FLT3 ligand as recognizing domain was constructed. The specific cytotoxicity against FLT3+ leukemia cell lines, primary AML cells, and normal hematopoietic progenitor stem cells (HPSCs) in vitro were evaluated. In addition, FLT3+ AML mouse model was used to assess the effect of FLT3L CAR-T therapy in vivo. Results FLT3L CAR-T cells could specifically kill FLT3+ leukemia cell lines and AML patients’ bone marrow mononuclear cells in vitro (with or without FLT3 mutation) and have more potent cytotoxicity to FLT3-ITD cells. In a human FLT3+ AML xenograft mouse model, FLT3L CAR-T cells could significantly prolong the survival of mice. Furthermore, it was found that FLT3L CAR-T cells could activate the FLT3/ERK signaling pathway of FLT3+ leukemia cells with wild-type FLT3; meanwhile, it had no inhibitory effects on the colony formation of CD34+ stem cells derived from normal human umbilical cord blood. Conclusions The ligand-based FLT3L CAR-T cells could be a promising strategy for FLT3+ AML treatment, especially those carried FLT3 mutation. Electronic supplementary material The online version of this article (10.1186/s13045-018-0603-7) contains supplementary material, which is available to authorized users.

Acute myeloid leukemia (AML) is a kind of a malignant hematologic tumor caused by uncontrolled repopulation of myeloid hematopoietic stem cells (HSCs). Current therapeutic effects for AML patients are unsatisfactory. In particular, relapsed and refractory AML still have a poor prognosis. T cells modified by chimeric antigen receptor (CAR) was an immunotherapeutic strategy for malignancies, which has a broad developing prospect. Most AML cells overexpress the myeloid antigen CD33. Therefore, CD33-specific CAR-T cells with different co-stimulators (CD28, 4-1BB, or both, referred to as CD33 28z.CAR-T cells, CD33 BBz.CAR-T cells, or CD33 28BBz.CAR-T cells, respectively) were developed to evaluate their efficacy against AML. The effectiveness of three types of CD33 CAR-T cells against AML was verified by specific killing effect to AML cells and prolonged survival of a xenograft mouse model. In terms of CAR-T cell efficacy, especially when transfused into human bodies, the persistence of T cells is also an important index, as it is closely associated with the long-term effect of CAR-T cells. Therefore, the characteristics of three types of CD33 CAR-T cells related to the persistence of T cells were examined. It was found that during expansion, CD33 BBz.CAR-T cells had an increased central memory compartment, while CD33 28z.CAR-T cells were predominantly effector memory T cells. In addition, CD33 28z.CAR-T cells were more inclined to become exhausted. The study suggests that incorporation of 4-1BB in CARs may endow T cells with long-lasting survival ability, thus improving the long-term anti-leukemia effect of CAR-T cells, especially when transfused to the human body.

Transcriptional factor Sox2 promotes tumor metastasis; however its regulatory effect on tumor-associated macrophages (TAMs, M2 phenotype) has not been defined. This study disclosed concomitant expression of TAMs marker-CD163 with SOX2 in human breast cancer and showed that Sox2 in breast cancer cells promotes recruitment of TAMs with altered expression of multiple chemokines, including MIP-1α, ICAM-1 etc. and activation of Stat3 and NF-κB signalings. In addition, TAMs rescued the compromised lung metastasis induced by Sox2 silencing in breast cancer cells. Together, this study documented that Sox2 plays an important role in recruiting TAMs and promotes tumor metastasis in a TAMs dependent manner.

The t(8;21)(q22;q22) translocation generated the fusion protein AML1-ETO. AML1-ETO recruits histone deacetylase (HDAC) complex via its ETO part to repress AML1-mediated transactivation. Our previous study demonstrated that HDAC inhibitor phenylbutyrate (PB) could induce AML1-ETO positive leukemia cell line Kasumi-1 cells to undergo differentiation and apoptosis accompanied by significant changes in gene expression profile. ZFP36L2 was one of the up-regulated genes in Kasumi-1 cells induced by PB treatment. In this study, ZFP36L2 was found to express at a lower level in acute myeloid leukemia (AML) patients with t(8;21) compared to AML patients without t(8;21). In order to investigate the correlation between the expression of ZFP36L2 and AML1 or AML1-ETO, the putative AML1 binding sites in the enhancer/promoter region of ZFP36L2 gene were predicted through the bioinformatics analysis. And the biological function of ZFP36L2 in leukemic cells was further investigated. The results demonstrated that AML1 could transactivate ZFP36L2 significantly by binding on specific site of the ZFP36L2 promoter sequence. And overexpression of ZFP36L2 in leukemia cells could inhibit the cell proliferation, promote cell-cycle arrest in G0/G1 phase and induce the cell apoptosis. In conclusion, ZFP36L2 could be transactivated by AML1, which subsequently induced cell-cycle arrest and apoptosis of leukemia cells.

Data from systemic clinical trials about chimeric antigen receptor-modified T cell therapy against CD19 (CD19 CAR) differ in CAR design, T-cell activation and transduction methods at different institutions. However, according to these clinical trials, the single-chain fragment variable (scFv) sequence specific for tumor antigen were mostly derived from the FMC63 or SJ25C1 clones. Our previous study showed that the CD19 CAR constructed in our laboratory derived from clone HI19α (HI19α-4-1BB-ζ CAR) was highly effective in preclinical models. Herein, we conducted a single-arm, phase I clinical trial to evaluate the safety and efficacy of HI19α-4-1BB-ζ CAR (CNCT19) in patients with relapsed/refractory acute lymphoblastic leukemia (R/R B-ALL). From November 2016 through December 2018, 20 R/R B-ALL patients were enrolled into this clinical trial. Complete remission (CR) or complete remission with incomplete count recovery (CRi) was achieved in 100% (18/18) of patients that could be evaluated on day 28 after infusion, which accounted for 90% of all 20 enrolled patients. After a median follow-up of 17.0 months (range, 0.2 - 19.8), the median overall survival (OS) for the entire cohort of patients was 9.6 months (95% CI 4.2 - 15.0), and was not reached for 14 patients bridge to allogeneic transplantation. The median relapse free survival (RFS) of all patients was 9.0 months (95% CI, 6.7 - 11.2). Two patients died within 28 days due to cytokine release syndrome (CRS), while other patients experienced controllable cytokine-release syndrome and neurotoxicity. In order to better understand the correlation between T cell subsets and long-term response, we consistently evaluated the T cell phenotype and expansion kinetics in peripheral blood after CART infusion. The results revealed that the percentage of CD8+ naïve T cells (TN) collected from peripheral blood 20min after CAR infusion, were significantly lower in patients who relapsed from CART therapy than patients with continues CR (p=0.003), while central memory T cells (TCM), effective memory T cells (TEM) and effector T cells (TE) had similar proliferation kinetics between these two groups. In addition, multivariate analysis indicated that low percentage of CD8+TN cells was an independent factor associated with shorter RFS (p=0.033, 95% CI 0.031-0.861). This report is the first trial to provide evidence that CNCT19, a CD19 CAR constructed of a new anti-CD19 chimeric antigen receptor HI19α, has potent antileukemia activities in patients with R/R B-ALL. Furthermore, our results indicate the phenotype and kinetics of T cells are possible biomarkers to predict the long-term prognosis of CART treatment. Disclosures Lv: Juventas Cell Therapy Ltd.: Employment.

As an important component of the tumor microenvironment, CD4+CD25+ Tregs reduce antitumor immunity, promote angiogenesis and metastasis in breast cancer. However, their function in regulating the “stemness” of tumor cells and the communication between Tregs and cancer stem cells (CSCs) remain elusive. Here, we disclose that the primarily cultured Tregs isolated from breast-tumor-bearing Foxp3-EGFP mouse upregulate the stemness property of breast cancer cells. Tregs increased the side-population and the Aldehyde dehydrogenase-bright population of mouse breast cancer cells, promoted their sphere formation in a paracrine manner, and enhanced the expression of stemness genes, such as Sox2 and so forth. In addition, Tregs increased tumorigenesis, metastasis, and chemoresistance of breast cancer cells. Furthermore, Sox2-overexpression tumor cells activated NF-κB-CCL1 signaling to recruit Tregs through reducing the binding of H3K27Me3 on promoter regions of p65 and Ccl1. These findings reveal the functional interaction between Tregs and CSCs and indicate that targeting on the communication between them is a promising strategy in breast cancer therapy.

SRY-related HMG-box gene 2 (SOX2) is one of the key regulatory genes that maintain the pluripotency and self-renewal properties in embryonic stem cells. Here we used immunohistochemistry to analyze the expression of SOX2 in human prostate tissues and found it contributed to tumorigenesis and correlated with histologic grade and Gleason score. We further investigated SOX2's function in cell growth and apoptosis process by using a human prostate cancer cell line DU145 with SOX2 overexpression or down-regulation. Cell cycle assay revealed that SOX2 promoted cell growth and increased the percentage of cells in S phase. In vitro and in vivo xenograft experiments in NOD/SCID mice further demonstrated that SOX2 increased the apoptosis-resistant properties of DU145 cells with decreased function of store-operated Ca(2+) entry and reduced expression of Orai1 at both mRNA and protein levels, suggesting a potential mechanism that contributes to the anti-apoptotic property of SOX2. To our knowledge, this study is the first to investigate SOX2's function in tumorigenesis and apoptosis of human prostate cancer and to elucidate its regulatory effect on the activity of store-operated Ca(2+) channels. Our results support the concept that SOX2 has the potential to be a significant marker to evaluate the progression of prostate cancer and serve as a potentially useful target for prostate cancer therapy.

Introduction: The pathogenesis of acute myeloid leukemia (AML) is always associated with chromosomal translocation, such as t(8;21)(q21;q22), which results in the formation of AML1-ETOfusion gene. The AML1-ETO fusion protein leads to hematopoietic differentiation blocked and leukemogenesis. How genomic abnormities such as chromosomal translocation influence the production and biological function of noncoding RNAs is relatively less studied. With the application of high-throughput sequencing and bioinformatics, it is increasing clear that the circular RNAs (circRNA) played important role in multiple biological processes. However, circRNA is rarely studied in hematopoietic malignancies. Here we assessed the roles of AML1-ETO related fusion circRNA (F-CircAE) in AML1-ETO leukemia. Methods: Total RNA was extracted from leukemia cell lines Kasumi-1 and SKNO-1, then treated with RNaseR or not. PCR assay with divergent primers was performed to identify F-CircAEs. The F-CircAE retroviral expression vector was constructed using Gibson Assembly Cloning Kit. NIH3T3 cells were transduced with retrovirus, the proliferation and foci formation were detected by crystal violet staining. F-CircAE-expressingNIH3T3 cells were injected subcutaneously into nude mice. Tumor growth was monitored at indicated time. To knock down the expression of F-CircAEs, the short hairpin double-stranded oligo targeting the back-splice junction of the F-CircAEs was inserted into the pLKO.1 lentiviral vector. Kasumi-1 cells were transduced with lentivirus, then the proliferation and cell cycle distribution of Kasumi-1 cells was measured by MTS assay and PI staining. Western blot was used to detect the cell cycle associated protein expression. RNA pulldown assay was performed with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the instructions. The bound proteins in the pulldown assay were analyzed by spectrum analysis. RNA-seq were performed using the Illumina platform. Sequencing reads were analyzed by a standard RNA-Seq pipeline. Gene Set Enrichment Analysis (GSEA) were used to analyze the pathway enrichment. Results: Several F-CircAEswere identified in AML1-ETO leukemia cell lines (Kasumi-1, SKNO-1) and t(8;21) patients' bone marrow mononuclear cells (BMMCs), but not in K562 cells and donors' BMMCs (Figure 1). To further validate F-CircAEs, RNaseR treatment was conducted and amplification products of circular RNAs were still detectable in AML1-ETO positive cells (Figure 1). Subsequently, Sanger sequencing of these amplification circular products revealed the head to tail sites of F-CircAE.To examine the function of F-CircAE, overexprseeion of F-CircAE in NIH3T3 cells resulted in increased proliferation compared with mutated F-CircAE (F-CircAE-mut) overexprssion or control group (Figure 2). Meanwhile, more foci were formed in the overexpressed F-CircAE group. In vivo,F-CircAE overexpression also promoted the growth of NIH3T3 cells in the injection site of nude mice. Furthermore, silencing of two F-CircAEs reduced the growth rates of Kasumi-1 cells (Figure 3)and induced an increase in G1 phase and a decrease in S and G2/M phase compared with scramble group. The protein levels of CDK2 and CyclinD1, which were the cell cycle regulatory components at G1 boundary, were reduced in F-CircAE knockdown groups compared with scramble group. To explore the mechanism by which F-CircAE promoted leukemia cells proliferation, RNA-pulldown assay was performed and more than 70 proteins were found to bind to F-CircAE. Based on the sequencing reads of RNA-seq, GSEA revealed several pathways were enriched following F-CircAE knockdown in Kasumi-1 cells. Conclusions: F-CircAEs were existed in t (8;21) leukemia cell lines and primary AML leukemia patients' BMMCs. F-CircAE could bind to proteins involved in the growth of leukemia cells. Discovery of F-CircAEs in AML1-ETO leukemia could make an immense progress in understanding their pathogenic mechanism, indicating new marker for diagnosis and therapy target. Disclosures No relevant conflicts of interest to declare.

Postoperative cognitive dysfunction (POCD) is a decline in cognitive function for weeks or months after surgery. It may affect the patients' length of hospital stay, quality of life, the rehabilitation process, and work performance. Prolonged POCD occurs frequently after cardiac surgery, and the risk of POCD increases with age. The pathophysiology of POCD has not been well understood. However, emerging evidences indicate that various inflammatory mediators are involved in the pathophysiology of POCD and inflammatory response may a potential pathogenic factor. The vagus nerve stimulation has been shown to decrease production and release of pro-inflammatory cytokines through the cholinergic anti-inflammatory pathway (CAP) in both animal model and human. Considering that the inflammation plays a definite role in the pathogenesis of POCD and the vagus nerve can mediate inflammation via CAP, we hypothesize that the transcutaneous vagus nerve stimulation may attenuate POCD by decreasing inflammatory response in elderly patients.

The limb remote ischaemia postconditioning (RIPOC), a newly conceived idea of applying brief limb ischaemic stimulus after onset of the myocardial ischaemia but prior to the reperfusion, can offer the possibility of applying this cardioprotective strategy to the patients with acute myocardial infraction. Although increasing numbers of circles and duration of limb ischaemia may augment the protective effect of the limb RIPOC, these ways are not feasible in a clinical setting. Morphine, a classic opioid drug, has been proven to enhance the cardioprotection obtained by the other interventions. Because both the RPIOC and opioid drugs produce the cardioprotection by multiple pathways, and there may be overlaps and cross talk in their mechanisms, we hypothesize that a combination of morphine and the limb RIPOC may produce an enhanced cardioprotection.

The importance of store-operated Ca2+ entry (SOCE) and the role of its key molecular regulators, STIM1 and ORAI1, in the development of cancer are emerging. Here, we report an unexpected dual function of SOCE in prostate cancer progression by revealing a decrease in the expression of STIM1 in human hyperplasia and tumor tissues of high histological grade and by demonstrating that STIM1 and ORAI1 inhibit cell growth by arresting the G0/G1 phase and enhancing cell senescence in human prostate cancer cells. In addition, STIM1 and ORAI1 inhibited NF-κB signaling and remodeled the tumor microenvironment by reducing the formation of M2 phenotype macrophages, possibly creating an unfavorable tumor microenvironment and inhibiting cancer development. However, STIM1 also promoted cell migration and the epithelial-to-mesenchymal transition by activating TGF-β, Snail and Wnt/β-Catenin pathways. Thus, our study revealed novel regulatory effects and the mechanisms by which STIM1 affects cell senescence, tumor migration and the tumor microenvironment, revealing that STIM1 has multiple functions in prostate cancer cells.

Background Recent study demonstrated the important contribution of SOX2 to tumorigenesis and metastasis properties of various types of cancers and strongly supported the concept that SOX2 can be used as an effective marker for diagnosis and predicting prognosis of cancer patients. However, our previous RNA-Seq results from human lung cancer cell line A549 showed that some oncogenes, including FOXA1 are negatively regulated by SOX2. Methods To further verify the transcriptional regulation effect of SOX2 on FOXA1 and elucidate its application in the diagnosis of human lung and breast cancer, we performed real-time RT-PCR and Western blotting to test the regulation effect of SOX2 on the expression of FOXA1 gene. OncoPrint analysis was used to reveal the alteration of SOX2 and FOXA1 genes in breast invasive carcinoma cases and lung squamous cell carcinoma cases from the Cancer Genome Atlas (TCGA) data portal. Immunohistochemistry staining was performed to test the expression of SOX2 and FOXA1 in human breast and lung carcinoma. Results The results showed that there is an inhibitory effect of SOX2 on the expression of FOXA1 gene. In addition, these two genes are altered in 5.8% of 484 breast invasive carcinoma cases and 46.4% of 179 lung squamous cell carcinoma cases from the Cancer Genome Atlas (TCGA) data portal, which showed an increased percentage of carcinoma cases when compared with single gene alteration. Immunohistochemistry staining of SOX2 and FOXA1 in human breast and lung carcinoma further revealed the mutual complementary effect of these two proteins in the diagnosis of carcinoma. Conclusions Our study revealed SOX2 as a negative upstream regulator for FOXA1 gene and demonstrated SOX2 and FOXA1 as effective dual markers in improving the diagnosis efficiency for human lung and breast tumor.

Introduction: TBLR1-RARα is the tenth fusion gene of acute promyelocytic leukemia (APL) first identified in a rare case of APL with t(3;17)(q26;q21) chromosomal translocation in our previous study. The characteristics of its basic structure and functions had been clarified in our previous study. In this study, we successfully established a novel TBLR1-RARα leukemia mouse model (TR mouse) which fully recapitulated the most relevant features of human APLs. The therapeutic effects of retinoic acid (ATRA), arsenic trioxide (As2O3), cytarabine (Ara-C) and histone deacetylase inhibitors (HDACi) on TR mice were examined. The differentially expressed genes (DEGs) between TR mice and normal mice were compared to explore the possible mechanisms and better therapeutic targets for this kind of APL. Methods: pMSCV-TBLR1-RARα-Flag-IRES-GFP (MSCV-TR) and pMSCV-IRES-GFP (vehicle) retroviral plasmids were constructed and transfected 293T packaging cells to produce retroviruses. Lin- cells from C57BL/6 mice bone marrow were purified and infected with MSCV-TR and vehicle retroviral supernatant. For in vitro assay, the GFP+ lin- cells sorted and incubated with or without different concentrations of ATRA were analyzed for the differentiation and proliferation capacity by cell morphology, myeloid markers (CD11b and GR-1) and colony formation assay. For the in vivo experiment, GFP+ lin- cells transfected with indicated retroviral vectors were injected intravenously to lethally irradiated C57BL/6 mice to establish an APL mouse model. Cell surface markers were analyzed by flow cytometry. In treatment assays, GFP+ spleen cells from TR leukemia mice were injected intravenously into recipient mice. The mice were randomly separated into groups and received different treatment with ATRA, As2O3, As2O3 in combination with ATRA, Ara-C, Ara-C in combination with ATRA, chidamide and NL101, respectively. The percentage of GFP+ cells in peripheral blood and body weight were measured dynamically. The survival time of every group was recorded and compared. RNA-seq assay was used to identify DEGs between TR mice and normal mice. Results: In vitro assays indicated that TBLR1-RARα could either block the differentiation of HSCs at a relatively early stage or enhanced the clonogenic potential of cells. The TBLR1-RARα leukemia mouse model was successfully established. During the ten-month observational period, 3 out of 15 mice transplanted with TBLR1-RARα expressing cells developed an APL-like disease. Development of leukemia was not observed in any of the mice in control group. All the leukemia mice had a body weight loss as well as splenomegaly and hepatomegaly. The phenotype analysis revealed that the progenitor markers Sca-1, CD34 and C-kit were positive, the myeloid lineage markers Gr-1 and CD11b were also positive, erythroid lineage marker Ter119 was weekly positive, but the lymphatic lineage marker B220, CD3,CD4 and CD8 were all negative. TR mice treated with 1.5-2.5 mg/kg ATRA alone or together with 2.0 mg/kg As2O3 didn't survive longer than that of control group, although in vitro differentiation experiment showed that the leukemia cells were sensitive to ATRA. Leukemic mice receiving Ara-C treatment had a much longer survive time. Surprisingly, HDAC inhibitors (12.5 and 25 mg/kg chidamide and 30 mg/kg NL-101) could significantly prolong the survival time of TR mice. Thousands of DEGs had been identified between TR mice and wild type mice, which were widely involved in multiple pathways and participated in various biological functions. Conclusion: The TBLR1-RARα leukemia mouse model was successfully established for the first time, and its main characteristics were clarified. Although the leukemia cells were sensitive to ATRA in vitro, TR mice didn't benefit from ATRA or As2O3 treatment in vivo. Besides Ara-C, HDAC inhibitors, such as chidamide and NL-101 exhibited potency therapeutic values for TR mice, which provided a new strategy for this kind of refractory APL. What' more, lots of genes that might be related with the process of leukemogenesis and new therapeutic targets for TR leukemia were identified. This model would serve as a versatile tool to study the mechanisms of leukemogenesis and help to design better strategies for APLs in further studies. Disclosures No relevant conflicts of interest to declare.

SOX2 was reported to promote metastasis in various tumor tissues; however the underlying mechanisms remain elusive. Here, we disclosed that SOX2 improves metastasis of breast and prostate cancer cells by promoting epithelial-to-mesenchymal transition (EMT) through WNT/β-catenin, but not TGF-β or Snail1 signaling. Dual luciferase assay and chromatin immunoprecipitation revealed activation and binding of SOX2 on promoter region of β-catenin. In addition, SOX2 affects the protein expression levels of DKK3, DVL1 and DVL3, which are regulators or downstream molecules of WNT signaling. Taken together, our findings demonstrated β-catenin as one of vital downstream molecules that mediate the EMT induced by SOX2.

Angiogenesis plays an important role during the development of tumor. Sox2 is one of the transcription factors involved in tumorigenesis and metastasis. However, its function during the process of tumor angiogenesis and lymphogenesis is still poorly understood. Here, we demonstrated that down-regulation of Sox2 significantly reduced the tumor angiogenesis and lymphogenesis as reflected by attenuated immunofluorescence staining of CD31 and LYVE-1 and improved prognosis in xenograft models of breast tumor. Furthermore, Sox2 also promoted the recruitment of bone marrow derived endothelial progenitor cells (MDEPC) to tumor microenvironment. Most importantly, we demonstrated that some of CD31+ and LYVE-1+ cells were derived from Sox2+ cancer cells and silencing of Sox2 could inhibit this transition. We revealed that Sox2 promoted the expression of LYVE-1, VEGFs and enhanced the phosphorylation of VEGFR2 in breast cancer. ChIP-seq and dual-luciferase assay further revealed the binding of Sox2 protein on proximate promoter region of VEGFB and LYVE1 to regulate their transcriptional activities, disclosing the underlying molecular mechanism on the regulatory effects of Sox2 on angiogenesis and lymphogenesis. Our study indicated that Sox2 improved tumor angiogenesis and lymphogenesis through regulating the related genes and unveiled a novel function of Sox2 in tumor progression. Citation Format: Rongrong Wang, Xuefei Li, Yingxi Xu, Tongchao Sun, Weijun Su, Chenhua Yu, Xiaoli Dong, Rong Xiang, Na Li. Sox2 enhances breast tumor angiogenesis by promoting the transition of cancer cell to CD31+ and LYVE-1+ cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2499.

Recent studies demonstrated that cancer stem cells (CSCs) have higher tumorigenesis properties than those of differentiated cancer cells and that transcriptional factor-SOX2 plays a vital role in maintaining the unique properties of CSCs; however, the function and underlying mechanism of SOX2 in carcinogenesis of lung cancer are still elusive. This study applied immunohistochemistry to analyze the expression of SOX2 in human lung tissues of normal individuals as well as patients with adenocarcinoma, squamous cell carcinoma, and large cell and small cell carcinoma and demonstrated specific overexpression of SOX2 in all types of lung cancer tissues. This finding supports the notion that SOX2 contributes to the tumorigenesis of lung cancer cells and can be used as a diagnostic probe. In addition, obviously higher expression of oncogenes c-MYC, WNT1, WNT2, and NOTCH1 was detected in side population (SP) cells than in non-side population (NSP) cells of human lung adenocarcinoma cell line-A549, revealing a possible mechanism for the tenacious tumorigenic potential of CSCs. To further elucidate the function of SOX2 in tumorigenesis of cancer cells, A549 cells were established with expression of luciferase and doxycycline-inducible shRNA targeting SOX2. We found silencing of SOX2 gene reduces the tumorigenic property of A549 cells with attenuated expression of c-MYC, WNT1, WNT2, and NOTCH1 in xenografted NOD/SCID mice. By using the RNA-Seq method, an additional 246 target cancer genes of SOX2 were revealed. These results present evidence that SOX2 may regulate the expression of oncogenes in CSCs to promote the development of human lung cancer.

The best treatment for myocardial infarction is to restore blood flow in the ischaemic region, though it will bring new myocardial damage known as myocardial ischaemia/reperfusion (I/R) injury. Both the ischaemia preconditioning and the ischaemia postcondioning have been shown to reduce the myocardial I/R injury, but their deficits restrict wide clinical availability. It has been demonstrated that inflammation plays a critical role in the I/R injury process. Also plasma levels of cytokines and inflammation response can be regulated by specifically augmenting cholinergic signaling via the efferent vagus nerve and alpha7 subunit-containing nicotinic acetylcholine receptor (alpha7nAChR). Because cholinergic modalities, acting through vagus nerve- and/or alpha7nAChR-mediated mechanism, have been confirmed to suppress excessive inflammation during the I/R injury in kidney, liver, lung and intestine, therefore, we hypothesize that cholinergic agonists may also provide a protection for the myocardial I/R injury.

Bortezomib (btz) is the major and potent agent in multiple myeloma (MM) treatment and intravenous (IV) injection is its standard administration route. However, peripheral neuropathy (PN) always limited the use of btz. Subcutaneous (SC) administration is an important alternative. The primary outcome of our previous study showed that SC administration of btz significantly decreased and delayed the development of PN in patients with newly diagnosed MM (NDMM) and relapse/refractory MM (RRMM). Here, we amplified the cohorts and extended follow up to further confirm our previous primary outcome in patients with NDMM. This retrospective, historical control study was undertaken at a single centre in China. Patients with NDMM were assigned to receive up to nine 21-day cycles of Btz based regimens, including PAd/VDd/BCd (btz 1.3 mg/m2 , on days 1, 4, 8 and 11; adriamycin 9 mg/m2 on days 1-4; or PLD 30mg/m2 on days 1, or CTX 500mg/m2 on days 1, 8, 15, and dexamethasone 20 mg/day on days 1, 2, 4, 5, 8, 9, 11 and 12). Btz was administered by SC injection or IV infusion. Then the efficacy and safety of SC versus IV btz were compared. 330 NDMM patients were received Btz based treatment. 151 received a median of 5 cycles of SC btz (SC group) from 2012.3 to 2014.12. 179 cases were treated with a median 3 cycles of IV btz (historical control IV group) from 2008.5 to 2012.2. The overall response rate (ORR, >1⁄4PR) was 97.4% and 97.2% in these two groups, with 37.7% and 36.3% achieved CR respectively, indication the efficacy was very comparable. SC group had better safety profile. Most of any grade adverse events (AE) were less common with SC than with IV administration, including thrombocytopenia, neutropenia, anaemia. The most importance was PN of any grade (32.1% vs 58.1%; p1⁄40$01) was significantly decreased with SC btz. Especially severe PN (grade 3/4) was also significantly decreased (5.3% vs 18.4%, p1⁄40.013). Notably, the incidence and seriousness of diarrhea were lower (any grade, 9.8% vs 15.7%, >1⁄4grade 3, 0.9% vs 4.7%, respectively), so were constipation, and intestinal obstruction, which may be related to the neuropathy toxicity of btz. The time of developing PN was delayed in SC group. Median dosage of btz when PN developed was 15.6 mg/m2 in SC group and 10.4mg/m2 in IV group. The efficacy of SC btz in patients with NDMM was comparable with standard IV administration. SC btz was locally well tolerated. It significantly decreased and delayed the development of PN. PO-143 Melphalan 140mg/m“2” significantly reduces 5 year progression free survival in multiple myeloma patients receiving a peripheral blood autologous stem cell transplant compared with melphalan 200mg/m“2” C. Attwood, E. Nicholson, R. Chakraverty, A. Fielding, P. Kottaridis, D. Hughes, A. Mehta, W. Stewart, C. Kyriakou, S. Worthington, S. Grace, A. Wechalekar, S. MacKinnon Royal Free Hospital, London

Recent study showed that Sox2 gene, a key stem cell gene that maintains the pluripotency properties of embryonic stem cell (ESC) and cancer stem cell (CSC) plays an important role in the tumorigenesis and metastasis process of various types of tumor, however its regulatory effect on tumor microenvironment (TME) is still elusive. In this study, we established a BALB/c mice model xenografted with a mice breast cancer cell line-4T1 with down-regulation of Sox2 gene (4T1-shRNA-Sox2). We found that down-regulation of Sox2 inhibits the growth of tumors with a modulated immunomicroenvironment, which was reflected by the increased number of CD3+CD8+ T cells and expression level of Th1 cytokines, such as TFN-γ and decreased number of Foxp3+ Tregs and expression level of Th2 cytokines such as IL4, IL6 and IL10. In addition, the reduced expression level of vascular endothelial cell marker CD31 and lymphatic vessel marker Lyve-1 was also revealed in the 4T1-shRNA-Sox2 xenografted tumors. We further demonstrated that Sox2 modulates the TME by recruiting Tregs. By using real-time RT-PCR, we found the cytokines contributing to recruitment of Tregs, such as CCL1, CCL22, CCL28 and Cxcl12 reduced after Sox2 downregulation. In addition, the mRNA expression level of transcriptional factor NF-κB and its co-transcriptional factor RelA which controlled the expression of CCL1, CCL22 and CCL28 were also reduced. Consequently, our study revealed the functional interaction between tumor cells and the immunocytes in TME by demonstrated that Sox2 gene in tumor cells recruited Tregs to influence the tumor microenvironment thus promotes the growth of tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 294. doi:1538-7445.AM2012-294

Ectopic expression of SOX2 has been reported in a variety of tumor tissues and contributes to cell proliferation and tumorigenesis by transcriptional regulation of the target genes; however the role of SOX2 in tumor metastasis and the underlying mechanisms remain largely elusive. Here we used immunohistochemistry to analyze the expression of SOX2 in the tissues of human breast and lymph nodes and found that SOX2 was highly expressed in breast cancer tissues and the metastasised lymph node, also the expression of SOX2 was closely correlated with the TNM stage and histological grade. We further demonstrated that SOX2 improved the migration and invasion properties of breast cancer and prostate cancer cells by facilitating the process of epithelial-mesenchymal transition (EMT). Investigation of the molecular mechanism revealed that SOX2 regulated EMT by β-catenin signaling pathway. Our findings prove that SOX2 plays a pivotal role in the metastasis of human breast cancer and prostate cancer and suggest that SOX2 has the potential to be developed as the diagnosis marker for breast and prostate cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1488. doi:10.1158/1538-7445.AM2011-1488