The nature of the signaling pathway(s) which initiate drug‐triggered apoptosis remains largely unknown and is of fundamental importance in understanding cell death induced by chemotherapeutic agents. Here we show that in the leukemic cell lines U937 and HL‐60, daunorubicin, at concentrations which trigger apoptosis, stimulated two distinct cycles of sphingomyelin hydrolysis (approximately 20% decrease at 1 microM) within 4–10 min and 60–75 min with concomitant ceramide generation. We demonstrate that the increase in ceramide levels, which precedes apoptosis, is mediated by a neutral sphingomyelinase and not by ceramide synthase. Indeed, potent ceramide synthase inhibitors such as fumonisin B1 did not affect daunorubicin‐triggered sphingomyelin hydrolysis, ceramide generation or apoptosis. In conclusion, we provide evidence that daunorubicin‐triggered apoptosis is mediated by a signaling pathway which is initiated by an early sphingomyelin‐derived ceramide production.

During the last several years, research has produced a significant amount of knowledge concerning the characteristics of human cd T lymphocytes. Findings regarding the immune functions of these cells, particularly their natural killer cell-like lytic activity against tumor cells, have raised expectations for the therapeutic applications of these cells for cancer. Pharmaceutical companies have produced selective agonists for these lymphocytes, and several teams have launched clinical trials of cd T cell-based cancer therapies. The findings from these studies include hematological malignancies (follicular lymphoma, multiple myeloma, acute and chronic myeloid leukemia), as well as solid tumors (renal cell, breast and prostate carcinomas), consisting of samples from more than 250 patients from Europe, Japan and the United States. The results of these pioneering studies are now available, and this short review summarizes the lessons learned and the role of cd T cell-based strategies in the current landscape of cancer immunotherapies.

The mammalian target of rapamycin (mTOR) is emerging as a promising target for antitumor therapy. However, the mechanism that contributes to its regulation in B lymphomas remains unknown. This study shows that in follicular lymphoma (FL) cells, mTOR is active because the cells displayed rapamycinsensitive phosphorylation of p70S6 kinase and 4E-BP1. Moreover, immunohistochemistry applied on lymph node tissue sections obtained from patients with FL revealed that, in most cases, p70S6 kinase was highly phosphorylated compared to normal tonsillar tissue. In FL cells, mTOR was under control of both phospholipase D (PLD) and phosphatidylinositol 3-kinase (PI3K). Moreover, we demonstrated that Syk plays a central role in mTOR activation because we found that both expression and activity are elevated compared to normal or chronic lymphocytic leukemia B cells. We also provide evidence that Syk operates through PLD-and PI3K-independent pathways. IntroductionNon-Hodgkin lymphoma (NHL) is the most commonly occurring malignant blood disease. Eighty-five percent of NHL belongs to the B lineage, and the most commonly occurring variety includes the diffuse large B-cell lymphoma (DLBCL). Follicular lymphoma (FL) is the second most common type of B-NHL and includes 35% to 40% of all adult lymphomas. FL is derived from germinal center follicle lymphoid cells. 1 FL is characterized by an indolent initial course, a favorable response to first-line therapy, and also recurrences followed by a refractory phase eventually complicated by histologic transformation. Although the prognosis of FL is variable, most patients with aggressive forms of FL (high FLIPI score) ultimately die from their disease and median survival is 5 to 8 years. 2 Despite the significant progress that has been made, mostly due to the introduction of rituximab combined with chemotherapy, further efforts are needed to identify new molecular targets in FL.In the vast majority of cases, FL cells display high expression of Bcl-2 as a consequence of t(14;18). 3,4 Based on the potent antiapoptotic properties of this protein, it has been proposed that not only is Bcl-2 a major, if not unique, causal factor for FL, but also that Bcl-2 overexpression represents a major obstacle for the eradication of tumor cells by chemotherapy, including that used in preparative regimens before stem cell transplantation. 5 However, approximately 10% to 15% of patients with FL are negative for Bcl-2. 6 Interestingly, clinical presentation, histopathologic findings, response to therapy, and clinical outcome in Bcl-2 ϩ patients are indistinguishable from those in Bcl-2 Ϫ FL patients. This observation suggests that, independently from Bcl-2, FL cells display alternative antiapoptotic pathways that ultimately interfere with the response to therapy. The recent identification of constitutively phosphorylated forms of AKT in FL histologic specimens supports this hypothesis. 7 The mammalian target of rapamycin, mTOR, is a 289-kDa evolutionarily conserved serine/threonine kinase that interacts and p...

In human blood, 1% to 5% of lymphocytes are ␥␦ T cells; they mostly express the ␥␦ T-cell receptor ( IntroductionThe success of therapeutic monoclonal antibodies (mAbs) in the treatment of cancer can be attributed to their multiple bioactivities. Their mechanism of action combines antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity, antibodydependent phagocytosis, direct cytotoxic activity, and inhibition of receptor signaling. 1 ADCC occurs when cytolytic effector cells expressing a receptor for the Fc region of the IgG class of antibodies (Fc␥ receptors) bind to antibodies on the surface of target cells. In humans, Fc␥ receptors comprise CD16 (Fc␥RIIIA-B), CD32 (Fc␥RIIA-C), and CD64 (Fc␥RI), which all bind the same region on IgG Fc but with low-to-medium (CD16, CD32) or high (CD64) affinities. 2 Several lines of evidence suggest that enhancing ADCC induced by therapeutic mAbs may directly improve their clinical efficacy. First, in mice bearing xenografted tumors, the efficacy of the therapeutic mAbs rituximab (RTX) and trastuzumab (TTZ) relies upon cell-surface expression of Fc␥R. 3 Second, ADCC is essential for the clinical efficacy of RTX in B-cell lymphoma patients and depends on the affinity of Fc␥RIIIA for the IgG. 4,5 Third, optimizing the affinity of RTX, TTZ, and alemtuzumab (ALZ) for Fc␥RIIIA increases their ADCC and their efficacy in preclinical and clinical studies. [6][7][8] Finally, recruitment and activation of additional cell effectors for ADCC might also enhance the cytolytic activity of anticancer mAbs. 9,10 The cytolytic effector cells involved in ADCC are CD16 ϩ (ie, Fc␥RIIIA)-positive natural killer (NK) cells and other CD8 ϩ cytolytic T lymphocytes, which release perforin through immunologic synapses to kill target cells. In addition, human CD4 Ϫ CD8 Ϫ ␥␦ T cells from peripheral blood might provide an important reservoir of cytolytic effector cells for ADCC. In most humans and nonhuman primate species, the majority of circulating ␥␦ T lymphocytes expresses the V␥9 T-cell receptor, with CD4 Ϫ CD8 Ϫ TCRV␥9 ϩ cells representing 1% to 3% of mononuclear cells. All these cells respond to stimulation with nonpeptide phosphoantigens (PAgs), which are small, phosphorylated metabolites produced by the cholesterol pathway in microbial pathogens and tumor cells. In addition to natural PAgs, the synthetic analog BrHPP 11 selectively stimulates TCRV␥9 ϩ ␥␦ T lymphocytes. PAg-stimulated ␥␦ T cells proliferate, secrete pro-inflammatory cytokines and chemokines, and, most importantly, kill leukemia, lymphoma, and carcinoma cells. 12,13 Several studies involving macaque monkeys 14,15 and clinical studies in cancer patients [16][17][18][19][20][21][22] have demonstrated in vivo the potential of PAg-activated TCRV␥9 ϩ ␥␦ T lymphocytes for cancer immunotherapy.The mechanism by which PAgs stimulate ␥␦ T cell-mediated cancer cell killing is unclear. The number of circulating ␥␦ T lymphocytes increases 50-to 100-fold in humans treated with BrHPP and IL2 (our unpublished observations...

The initiation of UV light-induced signaling in mammalian cells is largely considered to be subsequent to DNA damage. Several studies have also described ceramide (CER), a lipid second messenger, as a major contributor in mediating UV light-induced c-Jun N-terminal kinase (JNK) activation and cell death. It is demonstrated here that UV-C light irradiation of U937 cells results in the activation and translocation of a Zn 2؉ -independent acid sphingomyelinase, leading to CER accumulation in raft microdomains. These CERenriched rafts aggregate and play a functional role in JNK activation. The observation that UV-C light also induced CER generation and the externalization of acid sphingomyelinase and JNK in human platelets conclusively rules out the involvement of a nuclear signal generated by DNA damage in the initiation of a UV light response, which is generated at the plasma membrane.

Rituximab is a chimeric human immunoglobulin G1 (IgG1) anti-CD20 monoclonal antibody with significant activity against CD20 ؉ malignant B cells. Rituximab is currently used with success in the treatment of B-cell-derived lymphoid neoplasias either alone or in combination with chemotherapy. However, the predominant mechanism by which rituximab exerts its antitumor properties in vivo remains unknown. In the present study, we demonstrate that in Daudi and RL B-lymphoma cells, rituximab (without cross-linking) used at the saturating dose of 10 g/mL induced moderate accumulation in G 1 phase, growth inhibition, and significant loss in clonogenic potential. However, in these cells, rituximab induced no apoptosis. Furthermore, we observed that treatment with rituximab resulted in a rapid and transient increase in acid-sphingomyelinase (A-SMase) activity and concomitant cellular ceramide (CER) generation in raft microdomains. We also observed that rituximab-treated cells externalized both A-SMase and CER that colocalized with the CD20 receptor. Finally, we present evidence that rituximab-induced growth inhibition may be mediated through a CER-triggered signaling pathway, leading to the induction of cell cycle-dependent kinase inhibitors such as p27(Kip1) through a mitogenactivated protein kinase (MAPK)-dependent

Follicular lymphomas (FLs) account for 35–40% of all adult lymphomas. Treatment typically involves chemotherapy combined with the anti-CD20 monoclonal antibody (MAb) rituximab (RTX). The development of the type II anti-CD20 MAb obinutuzumab (GA101) aims to further improve treatment. Here, using FL cells we show that RTX and GA101 display a similar activity on RL cells cultured in 2D. However, 2D culture cannot mimic tumor spatial organization and conventional 2D models may not reflect the effects of antibodies as they occur in vivo. Thus, we created a non-Hodgkin's lymphoma (NHL) 3D culture system, termed multicellular aggregates of lymphoma cells (MALC), and used it to compare RTX and GA101 activity. Our results show that both antibodies display greater activity towards FL cells in 3D culture compared with 2D culture. Moreover, we observed that in the 3D model GA101 was more effective than RTX both in inhibiting MALC growth through induction of (lysosomal) cell death and senescence and in inhibiting intracellular signaling pathways, such as mammalian target of rapamycin, Akt, PLCgamma (Phospholipase C gamma) and Syk. Altogether, our study demonstrates that spatial organization strongly influences the response to antibody treatment, supporting the use of 3D models for the testing of therapeutic agents in NHL.

Immune checkpoint blockade therapeutics, notably antibodies targeting the programmed death 1 (PD-1) receptor and its PD-L1 and PD-L2 ligands, are currently revolutionizing the treatment of cancer. For a sizeable fraction of patients with melanoma, lung, kidney and several other solid cancers, monoclonal antibodies that neutralize the interactions of the PD-1/PD-L1 complex allow the reconstitution of long-lasting antitumor immunity. In hematological malignancies this novel therapeutic strategy is far less documented, although promising clinical responses have been seen in refractory and relapsed Hodgkin lymphoma patients. This review describes our current knowledge of PD-1 and PD-L1 expression, as reported by immunohistochemical staining in both non-Hodgkin lymphoma cells and their surrounding immune cells. Here, we discuss the multiple intrinsic and extrinsic mechanisms by which both T and B cell lymphomas up-regulate the PD-1/PD-L1 axis, and review current knowledge about the prognostic significance of its immunohistochemical detection. This body of literature establishes the cell surface expression of PD-1/PD-L1 as a critical determinant for the identification of non-Hodgkin lymphoma patients eligible for immune checkpoint blockade therapies.