Bin Meng

Activation of the STAT3 Signaling Pathway Is Associated With Poor Survival in Diffuse Large B-Cell Lymphoma Treated With R-CHOP

PURPOSE We previously reported that constitutive STAT3 activation is a prominent feature of the activated B-cell subtype of diffuse large B-cell lymphomas (ABC-DLBCL). In this study, we investigated whether STAT3 activation can risk stratify patients with DLBCL. PATIENTS AND METHODS By an immunohistochemical method, we investigated phosphotyrosine STAT3 (PY-STAT3) expression from 185 patients with DLBCL treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). Cell line-based siRNA experiments were also performed to generate an 11-gene, PY-STAT3 activation signature, which was used to study a previously published cohort of 222 patients with DLBCL. The STAT3 activation status determined by these two methods and by STAT3 mRNA levels were then correlated with survival. RESULTS PY-STAT3 was detected in 37% of DLBCL and enriched in ABC-DLBCL cases (P = .03). PY-STAT3 positivity significantly correlated with poor overall survival (OS; P = .01) and event-free survival (EFS; P = .006). Similar observations were made for high levels of STAT3 mRNA. In multivariable analysis, PY-STAT3 status (P = .02), International Prognostic Index (P = .02), and BCL2 expression (P = .046) were independent prognosticators of OS in this cohort. Among the cell-of-origin subgroups, PY-STAT3 was associated with poor EFS among non-germinal center B-cell DLBCL cases only (P = .027). Similarly, the 11-gene STAT3 activation signature correlated with poor survival in the entire DLBCL cohort (OS, P < .001; EFS, P < .001) as well as the ABC-DLBCL subgroup (OS, P = .029; EFS, P = .025). CONCLUSION STAT3 activation correlated with poor survival in patients with DLBCL treated with R-CHOP, especially those with tumors of the ABC-DLBCL subtype.

Genetic basis of PD-L1 overexpression in diffuse large B-cell lymphomas

Diffuse large B-cell lymphoma (DLBCL) is one of the most common and aggressive types of B-cell lymphoma. Deregulation of proto-oncogene expression after a translocation, most notably to the immunoglobulin heavy-chain locus (IGH), is one of the hallmarks of DLBCL. Using whole-genome sequencing analysis, we have identified the PD-L1/PD-L2 locus as a recurrent translocation partner for IGH in DLBCL. PIM1 and TP63 were also identified as novel translocation partners for PD-L1/PD-L2 Fluorescence in situ hybridization was furthermore used to rapidly screen an expanded DLBCL cohort. Collectively, a subset of samples was found to be affected by gains (12%), amplifications (3%), and translocations (4%) of the PD-L1/PD-L2 locus. RNA sequencing data coupled with immunohistochemistry revealed that these cytogenetic alterations correlated with increased expression of PD-L1 but not of PD-L2 Moreover, cytogenetic alterations affecting the PD-L1/PD-L2 locus were more frequently observed in the non-germinal center B cell-like (non-GCB) subtype of DLBCL. These findings demonstrate the genetic basis of PD-L1 overexpression in DLBCL and suggest that treatments targeting the PD-1-PD-L1/PD-L2 axis might benefit DLBCL patients, especially those belonging to the more aggressive non-GCB subtype.

Co-expression of PD-L1 and p-AKT is associated with poor prognosis in diffuse large B-cell lymphoma via PD-1/PD-L1 axis activating intracellular AKT/mTOR pathway in tumor cells

Programmed death-1 (PD-1) /programmed death-ligand 1 (PD-L1) engagement usually leads to diminished antitumor T-cell responses, which mediates the immune escape of tumor cells. However, little is known whether PD-1/PD-L1 could directly activates intracellular oncogenic signaling pathways in tumor cells. The purpose of this study is to investigate whether intracellular AKT/mTOR signaling could be directly activated by PD-1/PD-L1 during the malignant progression in diffuse large B-cell lymphoma (DLBCL). Detection of the expression of PD-L1 and p-AKT by immunohistochemistry (IHC) showed that both proteins were overexpressed in 54% and 48% DLBCL cases, respectively. Spearman test showed that PD-L1 expression was correlated with p-AKT expression (R=0.244, χ2=5.962; P=0.017) and the expression of PD-L1 and p-AKT were also correlated with clinic-pathological characteristics. In addition, survival analysis showed that DLBCL patients who co-expressed PD-L1 and p-AKT had significantly poorer outcome than patients with single positive or both negative expression (P<0.05). In vitro, total PD-L1 and membrane PD-L1 (mPD-L1) proteins were overexpressed in five DLBCL cell lines by western blot and flow cytometry. We observed that AKT/mTOR pathway was activated in DLBCL cells after stimulated with human recombination PD-1/Fc. Taken together, these results suggested that the combination of PD-1/PD-L1 antibodies and AKT/mTOR inhibitor might be a promising and novel therapeutic approach for DLBCL in the future.

Inhibition of 4EBP phosphorylation mediates the cytotoxic effect of mechanistic target of rapamycin kinase inhibitors in aggressive B-cell lymphomas

Mechanistic target of rapamycin (mTOR) complex 1 is a central integrator of nutrient and growth factor inputs that controls cell growth in eukaryotes. The second generation of mTOR kinase inhibitors (TORKi), directly targeting the mTOR catalytic site, are more effective than rapamycin and its analogs in cancer treatment, particularly in inducing apoptosis. However, the mechanism underlying the cytotoxic effect of TORKi remains elusive. Herein, we demonstrate that TORKi-induced apoptosis is predominantly dependent on the loss of mTOR complex 1-mediated 4EBP activation. Knocking out RICTOR, a key component of mTOR complex 2, or inhibiting p70S6K has little effect on TORKi-induced apoptosis. Conversely, increasing the eIF4E:4EBP ratio by either overexpressing eIF4E or knocking out 4EBP1/2 protects lymphoma cells from TORKi-induced cytotoxicity. Furthermore, downregulation of MCL1 expression plays an important role in TORKi-induced apoptosis, whereas BCL-2 overexpression confers resistance to TORKi treatment. We further show that the therapeutic effect of TORKi in aggressive B-cell lymphomas can be predicted by BH3 profiling, and improved by combining it with pro-apoptotic drugs, especially BCL-2 inhibitors, both in vitro and in vivo. Taken together, the study herein provides mechanistic insight into TORKi cytotoxicity and identified a potential way to optimize its efficacy in the clinical treatment of aggressive B-cell lymphoma.

Genetic landscape of hepatitis B virus–associated diffuse large B-cell lymphoma

Hepatitis B virus (HBV) infection is endemic in some parts of Asia, Africa, and South America and remains to be a significant public health problem in these areas. It is known as a leading risk factor for the development of hepatocellular carcinoma, but epidemiological studies have also shown that the infection may increase the incidence of several types of B-cell lymphoma. Here, by characterizing altogether 275 Chinese diffuse large B-cell lymphoma (DLBCL) patients, we showed that patients with concomitant HBV infection (surface antigen positive [HBsAg+]) are characterized by a younger age, a more advanced disease stage at diagnosis, and reduced overall survival. Furthermore, by whole-genome/exome sequencing of 96 tumors and the respective peripheral blood samples and targeted sequencing of 179 tumors from these patients, we observed an enhanced rate of mutagenesis and a distinct set of mutation targets in HBsAg+ DLBCL genomes, which could be partially explained by the activities of APOBEC and activation-induced cytidine deaminase. By transcriptome analysis, we further showed that the HBV-associated gene expression signature is contributed by the enrichment of genes regulated by BCL6, FOXO1, and ZFP36L1. Finally, by analysis of immunoglobulin heavy chain gene sequences, we showed that an antigen-independent mechanism, rather than a chronic antigenic simulation model, is favored in HBV-related lymphomagenesis. Taken together, we present the first comprehensive genomic and transcriptomic study that suggests a link between HBV infection and B-cell malignancy. The genetic alterations identified in this study may also provide opportunities for development of novel therapeutic strategies.

Synergistic antitumor effect of histone deacetylase inhibitor and Doxorubicin in peripheral T-cell lymphoma

Chidamide (CS055) is a new and highly selective histone deacetylase inhibitor displaying significant single-agent activity in peripheral T-cell lymphoma (PTCL). But there is little known the synergistic effect between CS055 and chemotherapy. The purpose of this study is to explore the synergistic effect and molecular mechanisms of CS055 combination with Doxorubicin in PTCL cells. We found that CS055 showed dose- and time-dependent inhibition effects on PTCL cell. Meanwhile, the synergistic effect was significantly observed after combination treatment with lower drug-concentration of CS055 and Doxorubicin. Lower drug-concentration of CS055 induced weak apoptosis in PTCL cells, but combination treatment with CS055 and Doxorubicin promoted more significant apoptosis. Combination treatment with CS055 and Doxorubicin significantly changed mitochondrial membrane potential and H3 acetylated level, resulting in up-regulating DNA damage protein p-γH2AX and apoptosis proteins including cleaved-caspase-3, cleaved-caspase-9 and cleaved-PARP, and down-regulating anti-apoptosis protein Bcl-2. In a word, Doxorubicin could increase the CS055-induced inhibition effects on PTCL cells, suggesting that CS055 combination with Doxorubicin or Doxorubicin-based chemotherapy drugs might be a new therapy approach for PTCL patients.

Prognostic Significance of BCL-2 and BCL-6 Expression in MYC-positive DLBCL

Micro‐Abstract: We investigated the significance of BCL‐2 and BCL‐6 expression in MYC+ diffuse large B‐cell lymphoma. Immunohistochemistry was performed to evaluate the expression of BCL‐2, BCL‐6, and MYC. BCL‐2 played a more important role than MYC in patients with double‐expression lymphoma. Besides, BCL‐6− expression might also be a negative prognostic factor for such patients. Background: Double‐expression lymphoma (DEL) is a rare subgroup of diffuse large B‐cell lymphoma (DLBCL), which has coexpression of MYC and BCL‐2. Coexpression of MYC and BCL‐2 is considered a prognostic marker portending poor outcomes. However, the prognostic effect of BCL‐2 and BCL‐6 expression in DLBCL remains controversial. Materials and Methods: Immunohistochemical staining was performed to detect MYC, BCL‐2 and BCL‐6 expression in 212 patients with newly diagnosed DLBCL and assess the prognostic effects of BCL‐2 and BCL‐6 expression. The DLBCL patients were treated with R‐CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine [Oncovin], prednisone)–like regimens. Results: Retrospective analysis revealed that BCL‐2+ and BCL‐2+/MYC+ were prognostic factors indicative of poor outcomes. Patients with BCL‐2+ and/or MYC+ expression had a poorer prognosis than that of patients with BCL‐2− and/or MYC− expression. Patients with BCL‐2+/MYC− expression showed a trend toward poorer survival than those with BCL‐2−/MYC+ expression, suggesting that BCL‐2 plays a more important role than MYC. Also, patients with BCL‐6−/MYC+ expression had poorer progression‐free survival than those with BCL‐6+/MYC+ expression. In addition, patients with BCL‐2+/MYC+/BCL‐6− expression had the worst prognosis, suggesting that BCL‐6− is a prognostic factor for poor outcomes for MYC+ DLBCL patients. Altogether, our findings have shown that BCL‐2 is an independent prognostic factor and possibly plays a more important role than MYC in MYC+ DLBCL patients. Furthermore, we found that BCL‐6− expression could also be a prognostic factor portending poor outcomes for MYC+ DLBCL patients.

Abstract 5568: Constitutively activation of STAT3 is a prognostic factor in activated B-cell subtype of diffuse large B-cell lymphoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We have previously reported aberrant activation of the signal transducer and activator of transcription 3 (STAT3) pathway promotes cell proliferation and survival in the activated B-cell-like (ABC) subtype, but not in the germinal center B-cell-like (GCB) subtype of diffuse large B-cell lymphoma (DLBCL). Herein, we investigated the effects of small molecular inhibitors of STAT3 in sensitizing DLBCL cell to chemotherapy drugs. ABC-DLBCL lines Ly3 and Pfeiffer were more resistant to vincristine-induced cell death than GCB-DLBCL cells Ly1 and Ly7 were. In ABC-DLBCL cells, constitutive activation of STAT3 provided survival signal and inhibited vincristine-induced apoptosis, while inhibition of STAT3 signaling abrogated IL-6 secretion and promoted cell apoptosis. STAT3 inhibitor and chemotherapy drugs synergistically induced cell apoptosis, with increased caspases 3/7 activity and PARP cleavage, in ABC-DLBCL cells but not in the GCB-DLBCL cells. The effect was correlated with reduced expression of MCL1 in these cells. To investigate the prognostic significance of STAT3 activation in DLBCL patients, we evaluated the levels of phospho-Tyr705-STAT3 (PY-STAT3) expression by immunohistochemistry in a cohort of 309 DLBCL patients treated with R-CHOP regimen. The ABC-DLBCL subgroup had significantly higher number of PY-STAT3 positive cases compared to the GCB subgroup (59.4% vs 36.2%; P = 0.003). Constitutive activation of STAT3 pathway has a significant impact on the overall-survival (OS) and event-free-survival (EFS) in the entire DLBCL cohort (OS, P = 0.010; EFS, P = 0.006) and in the ABC subgroup (OS, P = 0.062; EFS, P=0.016). Moreover, we constructed a subgroup of genes representing STAT3 activation from the gene expression profiling data of STAT3-siRNA treated ABC cell lines and primary DLBCL samples. Attributions of these genes include repression with the STAT3 knockdown in DLBCL cell lines, overexpression in the PY-STAT3-positive DLBCL tumors, and containing one or more STAT3 binding motif within their promoter regions. Eleven genes were obtained when trained this gene subgroup with patient survival through a semi-supervised algorithm. Averaged expression of the eleven genes predicted the OS (P < 0.001) and EFS (P < 0.001) in the entire DLBCL cohort, as well as in the ABC-DLBCL subgroup (OS, P = 0.029; EFS, P = 0.025). In conclusion, our study demonstrated that STAT3 activation is a significant prognostic factor in DLBCL and suggested that STAT3 may be a new therapeutic target in this aggressive malignancy. 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 5568. doi:1538-7445.AM2012-5568