Summary Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative, and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells, and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes.In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent nonneoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor.Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

miRNAs are small RNA molecules binding to partially complementary sites in the 3'-UTR of target transcripts and repressing their expression. miRNAs orchestrate multiple cellular functions and play critical roles in cell differentiation and cancer development. We analyzed miRNA profiles in B-cell subsets during peripheral B-cell differentiation as well as in diffuse large B-cell lymphoma (DLBCL) cells. Our results show temporal changes in the miRNA expression during B-cell differentiation with a highly unique miRNA profile in germinal center (GC) lymphocytes. We provide experimental evidence that these changes may be physiologically relevant by demonstrating that GC-enriched hsa-miR-125b down-regulates the expression of IRF4 and PRDM1/BLIMP1, and memory B cell-enriched hsa-miR-223 down-regulates the expression of LMO2. We further demonstrate that although an important component of the biology of a malignant cell is inherited from its nontransformed cellular progenitor-GC centroblasts-aberrant miRNA expression is acquired upon cell transformation. A 9-miRNA signature was identified that could precisely differentiate the 2 major subtypes of DLBCL. Finally, expression of some of the miRNAs in this signature is correlated with clinical outcome of uniformly treated DLBCL patients.

Purpose Diffuse large B-cell lymphoma (DLBCL) heterogeneity has prompted investigations for new biomarkers that can accurately predict survival. A previously reported 6-gene model combined with the international prognostic index (IPI) could predict patients’ outcome. However, even these predictors are not capable of unambiguously identifying outcome, suggesting that additional biomarkers might improve their predictive power. Experimental Design We studied expression of 11 microRNAs that had previously been reported to have variable expression in DLBCL tumors. We measured the expression of each microRNA by quantitative real-time polymerase-chain-reaction analyses in 176 samples from uniformly treated DLBCL patients and correlated the results to survival. Results In a univariate analysis, the expression of miR-18a correlated with overall survival (OS), whereas the expression of miR-181a and miR-222 correlated with progression-free survival (PFS). A multivariate Cox regression analysis including the IPI, the 6-gene model-derived Mortality Predictor Score and expression the of miR-18a, miR-181a, and miR-222, revealed that all variables were independent predictors of survival except the expression of miR-222 for OS and the expression of miR-18a for PFS. Conclusion The expression of specific miRNAs may be useful for DLBCL survival prediction and their role in the pathogenesis of this disease should be examined further.

IntroductionInterleukin-21 (IL-21) is a member of type I cytokine family that uses the shared ␥-common receptor chain for signaling. 1,2 It is predominantly secreted by activated CD4 ϩ T and natural killer (NK) T cells and induces pleiotropic effects on the immune system by regulating functions of T, B, NK, and myeloid cells. 1,3,4 The IL-21 receptor (IL-21R) has been reported to be present on almost all mature lymphocytes, with the highest expression on activated B cells. 5,6 The nature of IL-21's effects on B cells depends on the organism, specific cellular context (eg, activation and developmental stages), and presence of costimulatory factors. 7,8 IL-21 increases growth and differentiation of murine B lymphocytes that received both B-cell receptor (BCR) and T-cell help mediating signals while inducing apoptosis in cells lacking the concomitant BCR activation. 5 Although there have been several reports of IL-21 inducing apoptosis in murine B cells, the effects of IL-21 on human nonneoplastic B cells have been confined to regulation of B-cell activation and differentiation. Specifically, IL-21 has been shown to costimulate human B-cell proliferation induced by anti-CD40 antibody, yet inhibit proliferation induced by IL-4 and BCR stimulation. 1,6 IL-21 was also reported to have a central role in the differentiation of human primary B cells into plasma cells 9 and in promoting class-switch recombination and secretion of immunoglobulin G (IgG) and IgA in postswitch IgM ϩ memory B cells. 10 Since IL-21 was shown to stimulate the immune system, its effects on some tumors have been explored. IL-21 was reported to have potent antitumor activity in a variety of solid tumor models in mice. 11 Because solid tumors do not express IL-21R, these effects are likely to be indirect and mediated by IL-21-induced terminal differentiation of NK cells, regulation of T-cell differentiation and proliferation, and induction of cytotoxic T-cell responses. 12 In contrast to indirect immune-mediating effects of IL-21 on solid tumors, IL-21 may have direct effects on IL-21R-expressing malignancies originating from B lymphocytes. It was reported that IL-21 enhanced growth of multiple myeloma (MM) cells 13 yet induced apoptosis in chronic lymphocytic leukemia (CLL) B cells. 14,15 Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin lymphoma, is characterized by heterogeneity in clinical course and response to therapy. With the recent introduction of the anti-CD20 antibody rituximab into clinical practice, the "gold standard" therapy of DLBCL has evolved to include rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone. This has resulted in significant improvement in patient outcome, with 5-year survival reaching 50% to 60%. However, a significant proportion of patients still succumb to DLBCL and an urgent need for new therapies exists.Because IL-21 may be proapoptotic for certain B cells, we have examined the direct effects of IL-21 on DLBCL cell lines and primary tumors. We show that IL-21R is ...

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme shown to negatively regulate multiple tyrosine phosphorylation-dependent signaling pathways. PTP1B can modulate cytokine signaling pathways by dephosphorylating JAK2, TYK2, and STAT5a/b. Herein, we report that phosphorylated STAT6 may serve as a cytoplasmic substrate for PTP1B. Overexpression of PTP1B led to STAT6 dephosphorylation and the suppression of STAT6 transcriptional activity, whereas PTP1B knockdown or deficiency augmented IL-4-induced STAT6 signaling. Pretreatment of these cells with the PTK inhibitor staurosporine led to sustained STAT6 phosphorylation consistent with STAT6 serving as a direct substrate of PTP1B. Furthermore, PTP1B-D181A "substrate-trapping" mutants formed stable complexes with phosphorylated STAT6 in a cellular context and endogenous PTP1B and STAT6 interacted in an interleukin 4 (IL-4)-inducible manner. We delineate a new negative regulatory loop of IL-4-JAK-STAT6 signaling. We demonstrate that IL-4 induces PTP1B mRNA expression in a phosphatidylinositol 3-kinase-dependent manner and enhances PTP1B protein stability to suppress IL-4-induced STAT6 signaling. Finally, we show that PTP1B expression may be preferentially elevated in activated B cell-like diffuse large B-cell lymphomas. These observations identify a novel regulatory loop for the regulation of IL-4-induced STAT6 signaling that may have important implications in both neoplastic and inflammatory processes. IntroductionInterleukin 4 (IL-4) is a type I cytokine that has an important role in the regulation of Th2 cells and B cells during an immune response. IL-4 regulates cellular differentiation, proliferation, and apoptosis and plays an important role in the pathogenesis of allergic and autoimmune diseases. 1,2 Furthermore, recent studies have suggested that IL-4 signaling may have an important role in neoplastic diseases. IL-4 may affect malignant cells and can elicit potent antitumor activity against carcinoma and lymphoma cell lines in vitro and in animal models. [3][4][5][6] We have recently demonstrated qualitatively different IL-4 effects on gene expression, cell proliferation, and intracellular signaling in germinal center B-cell (GCB)-like versus activated B-cell (ABC)-like diffuse large B-cell lymphomas (DLBCLs). 7 Further, our preliminary data have suggested that IL-4 may enhance chemotherapy and complementdependent rituximab-mediated cytotoxicities in GCB-like but not in ABC-like DLBCLs. 8 The pleiotropic but specific effects of IL-4 on different cell types result from the activation of distinct signaling pathways that are tightly regulated. 1 IL-4 signaling is initiated when the cytokine binds its cell surface receptor activating receptor-associated Janusactivated protein kinases (JAKs) that phosphorylate specific tyrosine residues in the IL-4R␣ chain. This is followed by recruitment and phosphorylation of signal transducer and activator of transcription 6 (STAT6) and insulin receptor substance-2 (IRS-2), and activation of phosphatidylin...

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease characterized by variable clinical outcomes. Outcome prediction at the time of diagnosis is of paramount importance. Previously, we constructed a 6-gene model for outcome prediction of DLBCL patients treated with anthracycline-based chemotherapies. However, the standard therapy has evolved into rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Herein, we evaluated the predictive power of a paraffin-based 6-gene model in R-CHOP-treated DLBCL patients. RNA was successfully extracted from 132 formalin-fixed paraffinembedded (FFPE) specimens. Expression of the 6 genes comprising the model was measured and the mortality predictor score was calculated for each patient. The mortality predictor score divided patients into low-risk (below median) and high-risk (above median) subgroups with significantly different overall survival (OS; P ‫؍‬ .002) and progression-free survival (PFS; P ‫؍‬ .038). The model also predicted OS and PFS when the mortality predictor score was considered as a continuous variable (P ‫؍‬ .002 and .010, respectively) and was independent of the IPI for prediction of OS (P ‫؍‬ .008). These findings demonstrate that the prognostic value of the 6-gene model remains significant in the era of R-CHOP treatment and that the model can be applied to routine FFPE tissue from initial diagnostic biopsies. (Blood. 2008;111:5509-5514)

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.