Xiaoxi Zhou

Lymphoma endothelium preferentially expresses Tim-3 and facilitates the progression of lymphoma by mediating immune evasion

Angiogenesis is increasingly recognized as an important prognosticator associated with the progression of lymphoma and as an attractive target for novel modalities. We report a previously unrecognized mechanism by which lymphoma endothelium facilitates the growth and dissemination of lymphoma by interacting with circulated T cells and suppresses the activation of CD4+ T cells. Global gene expression profiles of microdissected endothelium from lymphoma and reactive lymph nodes revealed that T cell immunoglobulin and mucin domain–containing molecule 3 (Tim-3) was preferentially expressed in lymphoma-derived endothelial cells (ECs). Clinically, the level of Tim-3 in B cell lymphoma endothelium was closely correlated to both dissemination and poor prognosis. In vitro, Tim-3+ ECs modulated T cell response to lymphoma surrogate antigens by suppressing activation of CD4+ T lymphocytes through the activation of the interleukin-6–STAT3 pathway, inhibiting Th1 polarization, and providing protective immunity. In a lymphoma mouse model, Tim-3–expressing ECs promoted the onset, growth, and dissemination of lymphoma by inhibiting activation of CD4+ T cells and Th1 polarization. Our findings strongly argue that the lymphoma endothelium is not only a vessel system but also a functional barrier facilitating the establishment of lymphoma immune tolerance. These findings highlight a novel molecular mechanism that is a potential target for enhancing the efficacy of tumor immunotherapy and controlling metastatic diseases.

Tim-3 Expression in Cervical Cancer Promotes Tumor Metastasis

Background T cell immunoglobulin mucin-3 (Tim-3) has been identified as a negative regulator of anti-tumor immunity. Recent studies highlight the important role of Tim-3 in the CD8+ T cell exhaustion that takes place in both human and animal cancer models. However, the nature of Tim-3 expression in the tumor cell and the mechanism by which it inhibits anti-tumor immunity are unclear. This present study aims to determine Tim-3 is expressed in cervical cancer cells and to evaluate the role of Tim-3 in cervical cancer progression. Methodology A total of 85 cervical tissue specimens including 43 human cervical cancer, 22 cervical intraepithelial neoplasia (CIN) and 20 chronic cervicitis were involved. Tim-3 expression in tumor cells was detected and was found to correlate with clinicopathological parameters. Meanwhile, expression of Tim-3 was assessed by RT-PCR, Western Blot and confocal microscopy in cervical cancer cell lines, HeLa and SiHa. The migration and invasion potential of Hela cells was evaluated after inhibiting Tim-3 expression by ADV-antisense Tim-3. Conclusions We found that Tim-3 was expressed at a higher level in the clinical cervical cancer cells compared to the CIN and chronic cervicitis controls. We supported this finding by confirming the presence of Tim-3 mRNA and protein in the cervical cell lines. Tim-3 expression in tumor cells correlated with clinicopathological parameters. Patients with high expression of Tim-3 had a significant metastatic potential, advanced cancer grades and shorter overall survival than those with lower expression. Multivariate analysis showed that Tim-3 expression was an independent factor for predicting the prognosis of cervical cancer. Significantly, down-regulating the expression of Tim-3 protein inhibited migration and invasion of Hela cells. Our study suggests that the expression of Tim-3 in tumor cells may be an independent prognostic factor for patients with cervical cancer. Moreover, Tim-3 expression may promote metastatic potential in cervical cancers.

Arsenic disulfide synergizes with the phosphoinositide 3-kinase inhibitor PI-103 to eradicate acute myeloid leukemia stem cells by inducing differentiation

Although dramatic clinical success has been achieved in acute promyelocytic leukemia (APL), the success of differentiating agents has not been reproduced in non-APL leukemia. A key barrier to the clinical success of arsenic is that it is not potent enough to achieve a clinical benefit at physiologically tolerable concentrations by targeting the leukemia cell differentiation pathway alone. We explored a novel combination approach to enhance the eradication of leukemia stem cells (LSCs) by arsenic in non-APL leukemia. In the present study, phosphatidylinositol 3-kinase /AKT/mammalian target of rapamycin (mTOR) phosphorylation was strengthened after As(2)S(2) exposure in leukemia cell lines and stem/progenitor cells, but not in cord blood mononuclear cells (CBMCs). propidium iodide-103, the dual PI3K/mTOR inhibitor, effectively inhibited the transient activation of the PI3K/AKT/mTOR pathway by As(2)S(2). The synergistic killing and differentiation induction effects on non-APL leukemia cells were examined both in vitro and in vivo. Eradication of non-APL LSCs was determined using the nonobese diabetic/severe combined immunodeficiency mouse model. We found that a combined As(2)S(2)/PI-103 treatment synergized strongly to kill non-APL leukemia cells and promote their differentiation in vitro. Furthermore, the combined As(2)S(2)/PI-103 treatment effectively reduced leukemia cell repopulation and eradicated non-APL LSCs partially via induction of differentiation while sparing normal hematopoietic stem cells. Taken together, these findings suggest that induction of the PI3K/AKT/mTOR pathway could provide a protective response to offset the antitumor efficacy of As(2)S(2). Targeting the PI3K/AKT/mTOR pathway in combination with As(2)S(2) could be exploited as a novel strategy to enhance the differentiation and killing of non-APL LSCs.

Transmembrane TNF-α preferentially expressed by leukemia stem cells and blasts is a potent target for antibody therapy

To design an effective antibody therapy to improve clinical outcomes in leukemia, the identification of novel cell surface antigens is needed. Herein, we demonstrate a role for transmembrane tumor necrosis factor-α (tmTNF-α) in leukemia. To characterize tmTNF-α expression in acute leukemia (AL), normal hematopoietic cells, and nonhematopoietic tissues, we used a monoclonal antibody, termed C1, which specifically recognizes the tmTNF-α domain. We found that tmTNF-α was preferentially expressed by AL and leukemia stem cells (LSCs). More abundant expression correlated with poor risk stratification, extramedullary infiltration, and adverse clinical parameters. Moreover, knockdown of tmTNF-α(+) expression rendered leukemia cells more sensitive to chemotherapy in vitro and delayed regeneration of leukemia in NOD-SCID mice. Targeting tmTNF-α by C1 resulted in leukemia cell killing via antibody-dependent cell-mediated and complement-dependent cytotoxicity in vitro and inhibited leukemia cell growth in vivo while simultaneously sparing normal hematopoietic cells. Notably, C1 administration impaired the regeneration of leukemia in secondary serial transplantation into NOD-SCID mice. In conclusion, tmTNF-α has a favorable AL- and LSC-associated expression profile and is important for the survival and proliferation of these cells. C1-mediated targeting shows potent anti-LSC activity, indicating that tmTNF-α represents a novel target antigen in AL.

Tumor necrosis factor α in the onset and progression of leukemia

Tumor necrosis factor alpha (TNF-α), originally described as an anti-neoplastic cytokine, has been found, in apparent contradiction to its name, to play an important role in promoting the development and progression of malignant disease. Targeting TNF-α with TNF antagonists has elicited an objective response in certain solid tumors in phase I and II clinical trials. This review focuses on the relationship of TNF-α expressed by leukemia cells and adverse clinical features of leukemia. TNF-α is involved in all steps of leukemogenesis, including cellular transformation, proliferation, angiogenesis, and extramedullary infiltration. TNF-α is also an important factor in the tumor microenvironment and assists leukemia cells in immune evasion, survival, and resistance to chemotherapy. TNF-α may be a potent target for leukemia therapy.

Discovery of chrysoeriol, a PI3K-AKT-mTOR pathway inhibitor with potent antitumor activity against human multiple myeloma cells in vitro

This study was designed to determine the impact of chrysoeriol on proliferation and cell cycle progression in the human multiple myeloma cell lines RPMI 8226 and KM3, and its related molecular mechanisms. Chryseoriol was identified by using the phosphorylated AKT-specific cytoblot high throughput assay. CCK-8 assay was employed to examine the growth inhibition rate and IC50 (48 h) in peripheral blood mononuclear cells (PBMNCs), RPMI 8226 and KM3 cells treated with chrysoeriol at various concentrations. Cells were labeled with 5–6-carboxyfluorescein diacetate succinimidyl ester (CFSE), and the proliferation dynamics was detected by flow cytometry and analyzed with ModFit software. The cell cycles of RPMI 8226 and KM3 cells were measured by flow cytometry when the IC50 concentration of chrysoeriol was adopted. The alterations in cell-cycle related proteins (Cyclin B1, Cyclin D1, p21) and proteins in PI3K-AKT-mTOR pathway were determined by Western blot analysis. The results showed the proliferation of multiple myeloma cells was significantly inhibited by chrysoeriol, resulting in cell cycle arrest in G2/M phase. Chrysoeriol could significantly reduce the expression of p-AKT (s473) and p-4eBP1 (t37/46) protein, meanwhile enhanced Cyclin B1 and p21 protein expression. Similar effects were not observed in PBMNCs from normal donors. It was concluded that chrysoeriol was a selective PI3K-AKT-mTOR pathway inhibitor. It restrained the proliferation of human multiple myeloma cells, but didn’t affect proliferation of PBMNCs from normal donors. It might exhibit the cell cycle regulatory effect via the inhibition of PI3K-AKT-mTOR signal pathway.SummaryThis study was designed to determine the impact of chrysoeriol on proliferation and cell cycle progression in the human multiple myeloma cell lines RPMI 8226 and KM3, and its related molecular mechanisms. Chryseoriol was identified by using the phosphorylated AKT-specific cytoblot high throughput assay. CCK-8 assay was employed to examine the growth inhibition rate and IC50 (48 h) in peripheral blood mononuclear cells (PBMNCs), RPMI 8226 and KM3 cells treated with chrysoeriol at various concentrations. Cells were labeled with 5–6-carboxyfluorescein diacetate succinimidyl ester (CFSE), and the proliferation dynamics was detected by flow cytometry and analyzed with ModFit software. The cell cycles of RPMI 8226 and KM3 cells were measured by flow cytometry when the IC50 concentration of chrysoeriol was adopted. The alterations in cell-cycle related proteins (Cyclin B1, Cyclin D1, p21) and proteins in PI3K-AKT-mTOR pathway were determined by Western blot analysis. The results showed the proliferation of multiple myeloma cells was significantly inhibited by chrysoeriol, resulting in cell cycle arrest in G2/M phase. Chrysoeriol could significantly reduce the expression of p-AKT (s473) and p-4eBP1 (t37/46) protein, meanwhile enhanced Cyclin B1 and p21 protein expression. Similar effects were not observed in PBMNCs from normal donors. It was concluded that chrysoeriol was a selective PI3K-AKT-mTOR pathway inhibitor. It restrained the proliferation of human multiple myeloma cells, but didn’t affect proliferation of PBMNCs from normal donors. It might exhibit the cell cycle regulatory effect via the inhibition of PI3K-AKT-mTOR signal pathway.

TALENs-mediated gene disruption of FLT3 in leukemia cells: Using genome-editing approach for exploring the molecular basis of gene abnormality.

Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA-seq. The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These inhibitory effects were maintained in vivo, improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations.

SIL-TAL1 rearrangement is related with poor outcome: a study from a Chinese institution.

SIL-TAL1 rearrangement is common in T-cell acute lymphoblastic leukemia (T-ALL), however its prognostic implication remains controversial. To investigate the clinical characteristics and outcome of this subtype in Chinese population, we systemically reviewed 62 patients with newly diagnosed T-ALL, including 15 patients with SIL-TAL1 rearrangement. We found that SIL-TAL1+ T-ALL was characterized by higher white blood cell count (P = 0.029) at diagnosis, predominant cortical T-ALL immunophenotype (P = 0.028) of the leukemic blasts, and a higher prevalence of tumor lysis syndrome (TLS, P<0.001) and disseminated intravascular coagulation (DIC, P<0.001), which led to a higher early mortality (P = 0.011). Compared with SIL-TAL1− patients, SIL-TAL1+ patients had shorter relapse free survival (P = 0.007) and overall survival (P = 0.002). Our NOD/SCID xenotransplantation model also demonstrated that SIL-TAL1+ mice models had earlier disease onset, higher leukemia cell load in peripheral blood and shorter overall survival (P<0.001). Moreover, the SIL-TAL1+ mice models exerted a tendency of TLS/DIC and seemed vulnerable towards chemotherapy, which further simulated our clinical settings. These data demonstrate that SIL-TAL1 rearrangement identifies a distinct subtype with inferior outcome which could allow for individual therapeutic stratification for T-ALL patients.

Inhibition of STAT3 activity re-activates anti-tumor immunity but fails to restore the immunogenicity of tumor cells in a B-cell lymphoma model

A large number of patients with advanced lymphoma become refractory or relapse after initial treatment due to the persistence of minimal residual disease. Ideal immunotherapy strategy for eradicating the minimal residual disease of lymphoma and preventing the tendency to relapse need to be developed. Here, we use a mice model mimicked the disease entities of aggressive B-cell lymphoma dynamically to analyze the host anti-lymphoma immunity during the progression of lymphoma. We have shown that STAT3 activity was gradually enhanced in host immune effector cells with the progression of lymphoma. Inhibition of the STAT3 activity with a small molecule inhibitor was able to effectively enhance the function of both host innate and adaptive immunity, and thereby delayed the progression of lymphoma. Despite the therapeutic benefits were achieved by using of the STAT3 inhibitor, disrupting of STAT3 pathway did not prevent the eventual development of lymphoma due to the presence of point mutation of β2M, which controls immune recognition by T cells. Our findings highlight the complexity of the mechanism of immune evasion; therefore a detailed analysis of genes involved in the immune recognition process should be essential before an elegant immunotherapy strategy could be conducted.

A single fusion signal for t(14;18)(q32;q21) translocation is present in both the follicular lymphoma and local endothelial cells.

SummaryHerein we reported a case of follicular lymphoma with 50.26% clonal malignant lymphocytes and 50% tumor cells positive for the immunoglobulin heavy chain gene and B-cell lymphoma 2 gene (IGH-BCL2). To determine whether endothelial cells (ECs) within the tumor share the feature of advanced malignancy, we isolated and purified the ECs from the tumor by using the immunomagnetic beads conjugated with a monoclonal antibody against CD34, a surface marker of ECs. Thereafter, we identified ECs according to their morphology and found that ECs presented consistently flat and elongated appearance with a lot of Weibel-Palade bodies in the cytoplasm. Results of flow cytometry confirmed that ECs isolated from the follicular lymphoma expressed high level of both vWF and CD34 and the purity of the ECs fraction was more than 90%. Additionally, we used FISH to check chromosomal aberration in the purified ECs and found that some of the ECs had only one fusion signal for the green IGH probe and the red BCL2 probe in contrast to typical t(14;18)(q32;q21) translocation with two fusion signals. This phenomenon was also observed in the tumor cells. It might be a different breakpoint of IGH in this case, which induced the loss of the fusion signal, indicating t(14;18)(q32;q21) translocation. The positive cells accounted for 18% of the isolated ECs from the tumor, indicating that a proportion of ECs from follicular lymphoma had the same chromosome aberration as the neoplastic cells.Herein we reported a case of follicular lymphoma with 50.26% clonal malignant lymphocytes and 50% tumor cells positive for the immunoglobulin heavy chain gene and B-cell lymphoma 2 gene (IGH-BCL2). To determine whether endothelial cells (ECs) within the tumor share the feature of advanced malignancy, we isolated and purified the ECs from the tumor by using the immunomagnetic beads conjugated with a monoclonal antibody against CD34, a surface marker of ECs. Thereafter, we identified ECs according to their morphology and found that ECs presented consistently flat and elongated appearance with a lot of Weibel-Palade bodies in the cytoplasm. Results of flow cytometry confirmed that ECs isolated from the follicular lymphoma expressed high level of both vWF and CD34 and the purity of the ECs fraction was more than 90%. Additionally, we used FISH to check chromosomal aberration in the purified ECs and found that some of the ECs had only one fusion signal for the green IGH probe and the red BCL2 probe in contrast to typical t(14;18)(q32;q21) translocation with two fusion signals. This phenomenon was also observed in the tumor cells. It might be a different breakpoint of IGH in this case, which induced the loss of the fusion signal, indicating t(14;18)(q32;q21) translocation. The positive cells accounted for 18% of the isolated ECs from the tumor, indicating that a proportion of ECs from follicular lymphoma had the same chromosome aberration as the neoplastic cells.