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In addition to intrinsic genomic and nongenomic alterations, tumor progression is also dependent on the tumor microenvironment (TME, mainly composed of the extracellular matrix (ECM), secreted factors, and bystander immune and stromal cells). In chronic lymphocytic leukemia (CLL), B cells have a defect in cell death; contact with the TME in secondary lymphoid organs dramatically increases the B cells’ survival via the activation of various molecular pathways, including the B cell receptor and CD40 signaling. Conversely, CLL cells increase the permissiveness of the TME by inducing changes in the ECM, secreted factors, and bystander cells. Recently, the extracellular vesicles (EVs) released into the TME have emerged as key arbiters of cross-talk with tumor cells. The EVs’ cargo can contain various bioactive substances (including metabolites, proteins, RNA, and DNA); upon delivery to target cells, these substances can induce intracellular signaling and drive tumor progression. Here, we review recent research on the biology of EVs in CLL. EVs have diagnostic/prognostic significance and clearly influence the clinical outcome of CLL; hence, from the perspective of blocking CLL-TME interactions, EVs are therapeutic targets. The identification of novel EV inhibitors might pave the way to the development of novel combination treatments for CLL and the optimization of currently available treatments (including immunotherapy).

Resistance to death is one of the hallmarks of human B cell malignancies and often contributes to the lack of a lasting response to today’s commonly used treatments. Drug discovery approaches designed to activate the death machinery have generated a large number of inhibitors of anti-apoptotic proteins from the B-cell lymphoma/leukemia 2 family and the B-cell receptor (BCR) signaling pathway. Orally administered small-molecule inhibitors of Bcl-2 protein and BCR partners (e.g., Bruton’s tyrosine kinase and phosphatidylinositol-3 kinase) have already been included (as monotherapies or combination therapies) in the standard of care for selected B cell malignancies. Agonistic monoclonal antibodies and their derivatives (antibody–drug conjugates, antibody–radioisotope conjugates, bispecific T cell engagers, and chimeric antigen receptor-modified T cells) targeting tumor-associated antigens (TAAs, such as CD19, CD20, CD22, and CD38) are indicated for treatment (as monotherapies or combination therapies) of patients with B cell tumors. However, given that some patients are either refractory to current therapies or relapse after treatment, novel therapeutic strategies are needed. Here, we review current strategies for managing B cell malignancies, with a focus on the ongoing clinical development of more effective, selective drugs targeting these molecules, as well as other TAAs and signaling proteins. The observed impact of metabolic reprogramming on B cell pathophysiology highlights the promise of targeting metabolic checkpoints in the treatment of these disorders.

While Waldenstrom macroglobulinemia (WM) is characterized by an almost unifying mutation in MYD88, clinical presentation at diagnosis and response to therapy can be widely different among WM patients. Current prognostic tools only partially address this clinical heterogeneity. Limited data compiling both molecular and cytogenetic information have been used in risk prognostication in WM. To investigate the clinical impact of genetic alterations in WM, we evaluated cytogenetic and molecular abnormalities by chromosome banding analyses (CBA), FISH and targeted NGS in a retrospective cohort of 239 WM patients, including 187 patients treated by first-line chemotherapy or immunochemotherapy. Most frequent mutations were identified in MYD88 (93%), CXCR4 (29%), MLL2 (11%), ARID1A (8%), TP53 (8%), CD79A/B (6%), TBL1XR1 (4%) and SPI1 (4%). The median number of cytogenetic abnormalities was two (range, 0-22). Main cytogenetic abnormalities were 6q deletion (del6q) (27%), trisomy 4 (tri4) (12%), tri18 (11%), del13q (11%), tri12 (7.5%) and del17p (7%). Complex karyotype (CK) was observed in 15% (n=31) of cases, including 5% (n=12) of highly CK (high-CK). TP53 abnormalities (TP53abn) were present in 15% of evaluable patients. TP53abn and del6q were associated with CK/high-CK (P

In order to optimize the potency of the first serum-stable peptide agonist of CD47 (PKHB1) in triggering regulated cell death of cancer cells, we designed a maturation process aimed to mimic the trimeric structure of the thrombospondin-1/CD47 binding epitope. For that purpose, an N-methylation scan of the PKHB1 sequence was realized to prevent peptide aggregation. Structural and pharmacological analyses were conducted in order to assess the conformational impact of these chemical modifications on the backbone structure and the biological activity. This structure–activity relationship study led to the discovery of a highly soluble N-methylated peptide that we termed PKT16. Afterward, this monomer was used for the design of a homotrimeric peptide mimic that we termed [PKT16]3, which proved to be 10-fold more potent than its monomeric counterpart. A pharmacological evaluation of [PKT16]3 in inducing cell death of adherent (A549) and nonadherent (MEC-1) cancer cell lines was also performed.

International audience; The resistance to apoptosis of chronic lymphocytic leukemia (CLL) cells partly results from the deregulated production of survival signals from leukemic cells. Despite the development of new therapies in CLL, drug resistance and disease relapse still occur. Recently, neutrophil gelatinase-associated lipocalin (NGAL), a secreted glycoprotein, has been suggested to have a critical role in the biology of tumors. Thus, we investigated the relevance of NGAL in CLL pathogenesis, analyzed the expression of its cellular receptor (NGAL-R) on malignant B cells and tested whether CLL cells are resistant to apoptosis through an autocrine process involving NGAL and NGAL-R. We observed that NGAL concentrations were elevated in the serum of CLL patients at diagnosis. After treatment (and regardless of the therapeutic regimen), serum NGAL levels normalized in CLL patients in remission but not in relapsed patients. In parallel, NGAL and NGAL-R were upregulated in leukemic cells from untreated CLL patients when compared to normal peripheral blood mononuclear cells (PBMCs), and returned to basal levels in PBMCs from patients in remission. Cultured CLL cells released endogenous NGAL. Anti-NGAL-R antibodies enhanced NGAL-R + leukemia cell death. Conversely, recombinant NGAL protected NGAL-R + CLL cells against apoptosis by activating a STAT3/Mcl-1 signaling pathway. Our results suggest that NGAL and NGAL-R, overexpressed in untreated CLL, participate in the deregulation of the apoptotic machinery in CLL cells, and may be potential therapeutic clues for CLL treatment.

Objectives Apoptosis-Inducing Factor (AIF) is a protein involved in mitochondrial electron transport chain assembly/stability and programmed cell death. The relevant role of this protein is underlined because mutations altering mitochondrial AIF properties result in acute pediatric mitochondriopathies and tumor metastasis. By generating an original AIF-deficient mouse strain, this study attempted to analyze, in a single paradigm, the cellular and developmental metabolic consequences of AIF loss and the subsequent oxidative phosphorylation (OXPHOS) dysfunction. Methods We developed a novel AIF-deficient mouse strain and assessed, using molecular and cell biology approaches, the cellular, embryonic, and adult mice phenotypic alterations. Additionally, we conducted ex vivo assays with primary and immortalized AIF knockout mouse embryonic fibroblasts (MEFs) to establish the cell death characteristics and the metabolic adaptive responses provoked by the mitochondrial electron transport chain (ETC) breakdown. Results AIF deficiency destabilized mitochondrial ETC and provoked supercomplex disorganization, mitochondrial transmembrane potential loss, and high generation of mitochondrial reactive oxygen species (ROS). AIF-/Y MEFs counterbalanced these OXPHOS alterations by mitochondrial network reorganization and a metabolic reprogramming toward anaerobic glycolysis illustrated by the AMPK phosphorylation at Thr172, the overexpression of the glucose transporter GLUT-4, the subsequent enhancement of glucose uptake, and the anaerobic lactate generation. A late phenotype was characterized by the activation of P53/P21-mediated senescence. Notably, approximately 2% of AIF-/Y MEFs diminished both mitochondrial mass and ROS levels and spontaneously proliferated. These cycling AIF-/Y MEFs were resistant to caspase-independent cell death inducers. The AIF-deficient mouse strain was embryonic lethal between E11.5 and E13.5 with energy loss, proliferation arrest, and increased apoptotic levels. Contrary to AIF-/Y MEFs, the AIF KO embryos were unable to reprogram their metabolism toward anaerobic glycolysis. Heterozygous AIF+/- females displayed progressive bone marrow, thymus, and spleen cellular loss. In addition, approximately 10% of AIF+/- females developed perinatal hydrocephaly characterized by brain development impairment, meningeal fibrosis, and medullar hemorrhages; those mice died 5 weeks after birth. AIF+/- with hydrocephaly exhibited loss of ciliated epithelium in the ependymal layer. This phenotype was triggered by the ROS excess. Accordingly, it was possible to diminish the occurrence of hydrocephalus AIF+/- females by supplying dams and newborns with an antioxidant in drinking water. Conclusions In a single knockout model and at 3 different levels (cell, embryo, and adult mice) we demonstrated that by controlling the mitochondrial OXPHOS/metabolism, AIF is a key factor regulating cell differentiation and fate. Additionally, by providing new insights into the pathological consequences of mitochondrial OXPHOS dysfunction, our new findings pave the way for novel pharmacological strategies., Highlights • Mitochondrial AIF loss triggers OXPHOS/metabolism impairment. • AIF-/Y MEFs reprogram their metabolism by enabling an AMPK/GLUT-4 glycolytic pathway. • OXPHOS dysfunction associated with AIF loss generates a P53/P21 senescent phenotype. • The lack of metabolic plasticity provokes embryonic lethality in the AIF KO animals. • AIF+/- females develop immune cell exhaustion and perinatal hydrocephaly.

International audience

Gain in the short arm of chromosome 2 (2p+) induces gene overexpression and drug resistance in chronic lymphocytic leukemia: analysis of the central role of XPO1

International audience; Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia in Western countries, is a very heterogeneous disease characterized by a peripheral accumulation of abnormal CD5+ B lymphocytes in the immune system. Despite new therapeutic developments, there remains an unmet medical need for CLL. Here, we demonstrate that the use of N-methylated thrombospondin-1 (TSP-1)-derived peptides is an efficient way to kill the malignant CLL cells, including those from high-risk individuals with poor clinical prognosis, del11q, del17p, 2p gain, or complex karyotype. PKT16, our hit N-methylated peptide, triggers the elimination of the leukemic cells, sparing the nontumor cells, including the hematopoietic precursors, and reduces the in vivo tumor burden of a CLL-xenograft mice model. A complementary analysis underscores the improved cytotoxic efficiency of PKT16 compared with the previously described TSP-1-derived probes, such as PKHB1. PKT16 elicits an original caspase-independent programmed necrotic mode of cell death, different from necroptosis or ferroptosis, implicating an intracellular Ca2+ deregulation that provokes mitochondrial damage, cell cycle arrest, and the specific death of the malignant CLL cells. The activation of the Gαi proteins and the subsequent drop of cyclic adenosine monophosphate levels and protein kinase A activity regulate this cytotoxic cascade. Remarkably, PKT16 induces the molecular hallmarks of immunogenic cell death, as defined by the calreticulin plasma membrane exposure and the release of adenosine triphosphate and high-mobility group box 1 protein from the dying CLL cells. Thus, PKT16 appears to be able to stimulate an anticancer in vivo immune response. Collectively, our results pave the way toward the development of an efficient strategy against CLL.

International audience; B-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessment of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex ({greater than or equal to}3 abnormalities) in 73% of the patients and highly complex (>5 abnormalities) in 45%. The most frequent chromosomal aberrations were translocations involving MYC [t(MYC)] (62%), deletion (del)17p (38%), trisomy (tri)18 (30%), del13q (29%), tri3 (24%), tri12 (24%), and del8p (23%). Twenty-six of the 34 patients (76%) exhibit MYC aberration, resulting from mutually exclusive translocations or gains. Whole-exome sequencing revealed frequent mutations in TP53, MYD88, BCOR, MYC, SF3B1, SETD2, CHD2, CXCR4, and BCLAF1 The majority of B-PLL used the IGHV3 or IGHV4 subgroups (89%), and displayed significantly mutated IGHV genes (79%). We identified three distinct cytogenetic risk groups: low-risk (no MYC aberration), intermediate-risk (MYC aberration but no del17p), and high-risk (MYC aberration and del17p) (p=.0006). In vitro drug response profiling revealed that the combination of a B-cell receptor or BCL2 inhibitor with OTX015 (a bromodomain and extra-terminal motif (BET) inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We conclude that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease.

International audience; T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin-1-derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first-described serum-stable CD47-agonist peptide) on CEM and MOLT-4 human cell lines (T-ALL) and on one T-murine tumor lymphoblast cell-line (L5178Y-R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non-tumor cells after CD47 activation. In vivo, we determined that PKHB1-treatment in mice bearing the L5178Y-R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage-associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high-mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase-independent and calcium-dependent cell death in leukemic cells while sparing non-tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.

Leukemia cells from patients with acute myeloid leukemia (AML) display high proliferative capacity and are resistant to death. Membrane-anchored aminopeptidase-N/CD13 is a potential drug target in AML. Clinical research efforts are currently focusing on targeted therapies that induce death in AML cells. We previously developed a non-cytotoxic APN/CD13 inhibitor based on flavone-8-acetic acid scaffold, the 2',3-dinitroflavone-8-acetic acid (1). In this context, among the variously substituted 113 compounds further synthesized and tested for evaluation of their effects on APN/CD13 activity, proliferation and survival in human AML U937 cells, eight flavonoid derivatives emerged: 2',3-dinitro-6-methoxy-flavone-8-acetic acid (2), four compounds (3–6) with the 3-chloro-2,3-dihydro-3-nitro-2-phenyl-4H-1-benzopyran-4-one structure, and three (7–9) with the 3-chloro-3,4-dihydro-4-hydroxy-3-nitro-2-phenyl-2H-1-benzopyran framework. Different structure-activity relationships were observed between APN/CD13 activity and growth/survival processes. We showed that compound 2, but not benzopyran derivatives 3–9, inhibited APN activity (although to a less degree than 1). Both 1 and 2 did not affect AML cell proliferation and survival, indicating that CD13’s APN activity is not required for these processes. In contrast, benzopyran compounds 3–9 inhibited in a concentration-dependent manner the growth of U937 cells by inducing death as evidenced by phosphatidylserine externalization. Cell death was associated with the presence of geminal nitro group and chlorine at the 3-position of the 2H-1-benzopyran scaffold. The presence of other substituents such as CH2COOH or CH2CH=CH2 groups at the 8-position, NO2 or I substituents at the 2'- or 3'-position, OCH3 or OCH2C6H5 groups at the 4'-position did not affect cell death. Importantly, the inhibitory effects evidenced with compounds 7–9 were not due to their potential decomposition into the corresponding (Z)-(2chloro-2-nitroethenyl)benzene and salicylaldehyde. Based on these preliminary data, the 3-chloro,3-nitro-2H-1-benzopyran derivatives could be classified as a new group of compounds with promising antitumor properties; this study therefore provides the opportunity to explore their potential efficiency in AML patients’ cells ex vivo.

Mitochondrial metabolism is a tightly regulated process that plays a central role throughout the lifespan of hematopoietic cells. Herein, we analyze the consequences of the mitochondrial oxidative phosphorylation (OXPHOS)/metabolism disorder associated with the cell-specific hematopoietic ablation of apoptosis-inducing factor (AIF). AIF-null (AIF− /Y ) mice developed pancytopenia that was associated with hypocellular bone marrow (BM) and thymus atrophy. Although myeloid cells were relatively spared, the B-cell and erythroid lineages were altered with increased frequencies of precursor B cells, pro-erythroblasts I, and basophilic erythroblasts II. T-cell populations were dramatically reduced with a thymopoiesis blockade at a double negative (DN) immature state, with DN1 accumulation and delayed DN2/DN3 and DN3/DN4 transitions. In BM cells, the OXPHOS/metabolism dysfunction provoked by the loss of AIF was counterbalanced by the augmentation of the mitochondrial biogenesis and a shift towards anaerobic glycolysis. Nevertheless, in a caspase-independent process, the resulting excess of reactive oxygen species compromised the viability of the hematopoietic stem cells (HSC) and progenitors. This led to the progressive exhaustion of the HSC pool, a reduced capacity of the BM progenitors to differentiate into colonies in methylcellulose assays, and the absence of cell-autonomous HSC repopulating potential in vivo. In contrast to BM cells, AIF− /Y thymocytes compensated for the OXPHOS breakdown by enhancing fatty acid β-oxidation. By over-expressing CPT1, ACADL and PDK4, three key enzymes facilitating fatty acid β-oxidation (e.g., palmitic acid assimilation), the AIF− /Y thymocytes retrieved the ATP levels of the AIF +/Y cells. As a consequence, it was possible to significantly reestablish AIF− /Y thymopoiesis in vivo by feeding the animals with a high-fat diet complemented with an antioxidant. Overall, our data reveal that the mitochondrial signals regulated by AIF are critical to hematopoietic decision-making. Emerging as a link between mitochondrial metabolism and hematopoietic cell fate, AIF-mediated OXPHOS regulation represents a target for the development of new immunomodulatory therapeutics.

Resumo Partindo do pressuposto de que subjetividades são produzidas a partir das dimensões histórica, política e social e baseados nas discussões de Judith Butler acerca da violência normativa, pretendemos neste artigo, diante de recortes da violência de Estado, refletir sobre os processos de subjetivação de jovens considerados em conflito com a lei. Sustentada nas práticas experienciadas na (des)construção e (des)continuidade de um ano de estágio obrigatório em uma Delegacia Especializada, a discussão articula os recortes da experiência com conceitos como enquadramento, segurança pública e polícia cidadã. Para a Psicologia é importante não só olhar para esses processos de subjetivação da juventude, mas também levantar a discussão da implicação tanto a partir da posição de representantes do Estado, quanto da posição como sujeitos e cidadãos. Concluímos, portanto, que o(a) psicólogo(a) que atua(m) na polícia deve(m) assumir um papel político de deslocar o enquadramento e legitimar uma polícia que também se implique nesse emaranhado de questões. Abstract Based on the assumption that subjectivities are produced from historical, political and social dimensions and in light of Judith Butler’s discussions about normative violence, we intend in this article, in the face of state violence fragments, to reflect upon the subjectification processes of young people considered to be in conflict with the law. Sustained in the practices experienced in the construction and continuity of a year of internship in a Specialized Police Station, the discussion articulates the experience fragments with concepts such as framing, public safety and citizen police. To Psychology, it is important not only to look at these subjectification processes of young people, but also to raise the discussion of implication from the position of representatives of the state, as well as the position of subjects and citizens. We conclude, therefore, that the psychologist who works in the police must assume a political role of displacing, framing and legitimizing a police that is also involved in this tangle of issues. Resumen Partiendo del presupuesto de que subjetividades se producen a partir de las dimensiones histórica, política y social y basándose en discusiones de Judith Butler acerca de la violencia normativa, pretendemos en este artículo, delante de recortes de la violencia de Estado, reflexionar sobre los procesos de subjetivación de jovenes considerados en conflicto con la ley. Sustentada en las prácticas experimentadas de (des)construcción y (des)continuidad de un año de prácticas curriculares en una Comisaría Especializada, la discusión articula los recortes de la experiencia con conceptos como encuadramiento, seguridad pública y policía ciudadana. Para la Psicología es importante no solo mirar para estos procesos de subjetivación de la juventud como también levantar la discusión sobre la implicación tanto a partir de la posición de representantes del Estado, cuanto de la posición como sujetos y ciudadanos. Concluimos, por lo tanto, que el(la) psicólogo(a) que actúa(n) en la policía debe(n) asumir un rol político de dislocar el encuadramiento y legitimar una policía que también se implique en este enmarañado de cuestiones.

The human Apoptosis Inducing Factor (hAIF) is a bifunctional NAD(P)H-dependent flavoreductase involved in both mitochondrial energy metabolism and caspase-independent cell death. Even though several studies indicate that both functions are redox controlled by NADH binding, the exact role of hAIF as a reductase in healthy mitochondria remains unknown. Upon reduction by NADH, hAIF dimerizes and produces very stable flavin/nicotinamide charge transfer complexes (CTC), by stacking of the oxidized nicotinamide moiety of the NAD(+) coenzyme against the re-face of the reduced flavin ring of its FAD cofactor. Such complexes are critical to restrict the hAIF efficiency as a reductase. The molecular basis of the hAIF reductase activity is here investigated by analyzing the role played by residues contributing to the interaction of the FAD isoalloxazine ring and of the nicotinamide moiety of NADH at the active site. Mutations at K177 and E314 produced drastic effects on the hAIF ability to retain the FAD cofactor, indicating that these residues are important to set up the holo-enzyme active site conformation. Characterization of P173G hAIF indicates that the stacking of P173 against the isoalloxazine ring is relevant to determine the flavin environment and to modulate the enzyme affinity for NADH. Finally, the properties of the F310G and H454S hAIF mutants indicate that these two positions contribute to form a compact active site essential for NADH binding, CTC stabilization, and NAD(+) affinity for the reduced state of hAIF. These features are key determinants of the particular behavior of hAIF as a NADH-dependent oxidoreductase.

Apoptosis is nowadays what comes first to your scientist mind when someone mentions cellular suicide. However this is not the sole form of programmed cell death and many other alternative or atypical pathways have now been described. These pathways are indeed rather preferred to apoptosis in some instances based on tissue origin, cell type or development stage of the target cell. In this review, we describe many different programmed cell death subtypes according to their characteristics. Discrete, brutal, final or singular cell death pathways all participate in the elimination of unwanted, damaged or dangerous cells in organisms hence contributing to our knowledge of this particular feature of living beings: dying! Through description of anoikis, necroptosis, entosis, netosis, pyroptosis or ferroptosis, we have no choice but to realize that programmed cell death comes in many flavors.

International audience; Abstract ImageThrombospondin-1 (TSP-1) is a glycoprotein considered as a key actor within the tumor microenvironment. Its binding to CD47, a cell surface receptor, triggers programmed cell death. Previous studies allowed the identification of 4N1K decapeptide derived from the TSP-1/CD47 binding epitope. Here, we demonstrate that this peptide is able to induce selective apoptosis of various cancer cell lines while sparing normal cells. A structure–activity relationship study led to the design of the first serum stable TSP-1 mimetic agonist peptide able to trigger selective programmed cell death (PCD) of at least lung, breast, and colorectal cancer cells. Altogether, these results will be of valuable interest for further investigation in the design of potent CD47 agonist peptides, opening new perspectives for the development of original anticancer therapies.

International audience; A growing body of evidence points towards smoking-related phenotypic differences in chronic obstructive pulmonary disease (COPD). As COPD is associated with systemic inflammation, we determined whether smoking status is related to serum levels of matrix metalloproteinase-9 (pro-and active MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) and the proMMP-9/NGAL complex in patients with COPD. Serum samples were collected in 100 stable-phase COPD patients (82 smokers, 18 never-smokers) and 28 healthy adults (21 smokers, 7 never-smokers). Serum levels of studied factors were measured in ELISA. Our data provide the first evidence of simultaneously elevated serum levels of MMP-9, NGAL and proMMP-9/NGAL in COPD smokers. While the triad discriminated between smokers and non-smokers in the COPD group, MMP-9 and proMMP-9/NGAL (but not NGAL) discriminated between smokers with and without COPD. Adjustment for age and smoking pack-years did not alter the findings. Serum MMP-9, NGAL and proMMP-9/NGAL levels were not correlated with the GOLD stage or FEV1 decline. Furthermore, serum levels of neutrophil elastase (NE) and MMP-3 (but not of IL-6 and MMP-12) were also higher in COPD smokers than in healthy smokers before and after adjustment for age and pack-years. Among COPD smokers, levels of MMP-9, NGAL and proMMP-9/NGAL were positively correlated with NE (P < 0.0001) but not with the remaining factors. Gelatin zymography detected proMMP-9 in serum samples of healthy and COPD smoking groups. Our results suggest that associated serum levels of proMMP-9, NGAL, proMMP-9/NGAL and NE may reflect the state of systemic inflammation in COPD related to cigarette smoking.

T cell memory is the hallmark of adaptive immunity. Central questions are to determine which cells among proliferating effector T cells will live beyond the crash of the immune response (IR) and develop into functional memory T cells. CD47, considered as a marker of self, is implicated in cell death, cell elimination, and in the inflammatory response. We report in this article that CD47 expression was transiently regulated on Ag-specific CD4 T cells, that is, from CD47high to CD47low to CD47high, during the course of the in vivo IR. Specifically, CD47high status marked central memory CD4 T cell precursors at an early time point of the IR. By contrast, cytokine production was a functional attribute restricted to CD47high, but not CD47low, polyclonal effector CD4 T cells during recall responses in an experimental model of chronic airway inflammatory disease. Passive transfer of CD47high, but not CD47low, CD4 T cells in nonlymphopenic naive mice generated long-lived memory T cells capable of anamnestic responses. We conclude that CD47high status on CD4 T cells identifies functional long-lived memory T cell progenitors.

Alkylating DNA-damage agents such as N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) trigger necroptosis, a newly defined form of programmed cell death (PCD) managed by receptor interacting protein kinases. This caspase-independent mode of cell death involves the sequential activation of poly(ADP-ribose) polymerase-1 (PARP-1), calpains, BAX and AIF, which redistributes from mitochondria to the nucleus to promote chromatinolysis. We have previously demonstrated that the BAX-mediated mitochondrial release of AIF is a critical step in MNNG-mediated necroptosis. However, the mechanism regulating BAX activation in this PCD is poorly understood. Employing mouse embryonic knockout cells, we reveal that BID controls BAX activation in AIF-mediated necroptosis. Indeed, BID is a link between calpains and BAX in this mode of cell death. Therefore, even if PARP-1 and calpains are activated after MNNG treatment, BID genetic ablation abolishes both BAX activation and necroptosis. These PCD defects are reversed by reintroducing the BID-wt cDNA into the BID(-/-) cells. We also demonstrate that, after MNNG treatment, BID is directly processed into tBID by calpains. In this way, calpain non-cleavable BID proteins (BID-G70A or BID-Δ68-71) are unable to promote BAX activation and necroptosis. Once processed, tBID localizes in the mitochondria of MNNG-treated cells, where it can facilitate BAX activation and PCD. Altogether, our data reveal that, as in caspase-dependent apoptosis, BH3-only proteins are key regulators of caspase-independent necroptosis.

B-PLL is defined by the presence of prolymphocytes in peripheral blood exceeding 55% of lymphoid cells. The diagnosis, mainly based on clinical and morphological data, can be difficult because of overlap with other B-cell malignancies. Because of the rarity of the disease, only case reports and small series describe its cytogenetic features. Few prognostic markers have been identified in this aggressive leukemia usually resistant to standard chemo-immuno therapy. We report here the cytogenetic and molecular findings in a large series of B-PLL. We also studied the in vitro response to novel targeted drugs on primary B-PLL cells. The study included 34 cases with a diagnosis of B-PLL validated by morphological review performed by three independent expert cytologists. The diagnosis of mantle cell lymphoma was excluded by karyotype (K) and FISH using CCND1, CCND2 and CCND3 probes. Median age at diagnosis was 72 years [46-88]. K was complex (≥3 abnormalities) in 73%, and highly complex (HCK≥5) in 45%. Combining K and FISH data, the most frequent chromosomal aberrations were: translocation targeting the MYC gene [t(MYC)] (21/34, 62%), 17p deletion including TP53 gene (13/34, 38%), trisomy 18/18q (10/33, 30%), 13q14 deletion (10/34, 29%), trisomy 3 (8/33, 24%), trisomy 12 (8/34, 24%) and 8p deletion (7/31, 23%). Whole-Exome Sequencing analysis of paired tumor-control DNA was performed in 16 patients. The most frequently mutated genes were TP53(6/16, 38%), associated with del17p in all, MYD88 (n=4), BCOR (n=4), MYC (n=3), SF3B1 (n=3), FAT1 (n=3), SETD2 (n=2), CHD2 (n=2), CXCR4 (n=2), BCLAF1 (n=2) and NFASC (n=2). Distribution of the chromosomal aberrations is shown in Fig 1. The main group of patients (21/34, 62%) had a t(MYC) that was associated with a higher % of prolymphocytes (86 vs 76, p=0.03), CD38 expression (90% vs 15%,p The median overall survival (OS) for the entire cohort was 126 months with a median follow-up time of 47 months [ 0.2-141]. We found MYC activation (translocation or gain) to be associated with a shorter OS (p=0.03). Regarding MYC and del17p, we identified 3 distinct cytogenetic prognostic groups, with significant differences in OS (p=0.0006) (Fig 2). The patients without MYC activation had the lower risk (n=8, median not reached). Patients with a MYC activation without del17p had an intermediate risk (n=18, 125 months). The highest risk group corresponded to patients with both MYC and TP53 aberrations (n=7, 11 months). We performed drug response profiling on primary B-PLL cells using the ATP-based CellTiter Glo kit (Promega) (n=5). We observed that after 48h of exposure to increased doses, response was heterogeneous, with a majority of samples resistant to fludarabine (n=3), ibrutinib (n=3), idelalisib (n=4), venetoclax (n=3) and OTX015 (n=4). Annexin/PI assays using flow cytometry showed that the induced cell death could be increased by combination of ibrutinib or venetoclax with OTX015 or JQ1, two BET protein inhibitors that target MYC signaling (n=1/2). In summary, B-PLL have complex and highly complex K, a high frequency of MYC activation by translocation or gain, frequent 17p deletion, and frequent mutations in MYC, TP53, BCOR, and MYD88 genes. We identified 3 prognostic subgroups according to MYC and 17p status. Patients with MYC activation + 17p deletion have the shorter OS, and should be considered as a high-risk "double-hit" subgroup. Our results show that cytogenetic analysis is a useful diagnostic tool in B-PLL that improves prognostic stratification. We recommend to perform K and FISH (MYC and TP53) analyses systematically when a B-PLL is suspected. Our in vitro data suggest that drugs targeting the BCR and BCL2 in combination with MYC inhibition may be a therapeutic option in some patients. Disclosures Baseggio: Takeda Oncology: Honoraria.

International audience; Human neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein present in a wide variety of tissues and cell types. NGAL exists as a 25 kDa monomer, a 46 kDa homodimer (the most abundant form in healthy subjects) and a 130 kDa disulfide-linked heterodimer bound to latent matrix metalloproteinase-9. Dysregulated expression of NGAL in human malignancies suggests its value as a clinical marker. A growing body of evidence is highlighting NGAL's paradoxical (i.e., both beneficial and detrimental) effects on cellular processes associated with tumor development (proliferation, survival, migration, invasion, and multidrug resistance). At least two distinct cell surface receptors are identified for NGAL. This review (i) summarizes our current knowledge of NGAL's expression profiles in solid tumors and leukemias, and (ii) critically evaluates the beneficial and detrimental activities of NGAL having been documented in a diverse range of cancer-derived cell lines. A better understanding of the causal relationships between NGAL dysregulation and tumor development will require a fine analysis of the molecular aspects and biological role(s) of NGAL both in primary tumors and at different stages of disease. Having an accurate picture of NGAL's contribution to tumor progression is a prerequisite for attempting to modulate this protein as a putative therapeutic target.

Caspase-independent programmed necrosis is a highly regulated cellular demise that displays morphological and biochemical necrotic hallmarks, such as an earlier permeability of the plasma membrane and lactate dehydrogenase (LDH) leakiness. This form of programmed cell death (PCD) is regulated by AIF, a FAD-dependent oxidoreductase, which is released from the mitochondria to the nucleus where it induces chromatin tcondensation and DNA fragmentation. Some years ago, it was established that the sequential activation of poly(ADP-ribose) polymerase- 1 (PARP-1), calpains and Bax regulates the mitochondrial AIF release associated to programmed necrosis. But, what happens when AIF is in the nucleus? How does this protein induce chromatinolysis and programmed necrosis? Recently, we have unraveled some of the mechanisms underlying the nuclear action of AIF in this type of caspase-independent cell death. Indeed, AIF plays a key role in programmed necrosis by its ability to organize a DNA-degrading complex with H2AX and Cyclophiline A (CypA). The AIF/H2AX link is indeed a critical event and explains the nuclear AIF apoptogenic action. In the present article, we outline the current knowledge on cell death by programmed necrosis and discuss the relevance of the AIF/H2AX/CypA DNA-degrading complex in the regulation of this original form of cell death.

International audience; Acetogenins of the Annonaceae are strong inhibitors of mitochondrial complex I but discrepancies in the structure/activity relationships pled the search for other targets within the whole cell proteome. Combining hemisynthetic work, Cu-catalyzed Huisgen cycloaddition and proteomic techniques we have identified new putative protein targets of squamocin ruling out the previously accepted 'complex I dogma'. These results give new insights into the mechanism of action of these potent neurotoxic molecules.

The past decades have been marked by spectacular progress towards understanding how dendritic cells (DCs) interact with T cells to elicit protective immune responses to fight infectious diseases and cancer. DCs that are lying at the interface between innate and adaptive immunity, are educated in peripheral tissues prior to their journey to the secondary lymphoid organs (SLO) whereby they dictate different classes of T cell responses. Uncontrolled or unwanted inflammatory responses are the price to pay to eliminate pathogens. However, if not self-limited, they may induce collateral damages that result in chronic inflammation often associated with autoimmune disorders. CD47 and its two ligands, i.e. thrombospondin 1 (TSP-1) and SIRP-α, were identified as a previously unappreciated inhibitory axis of DC and T cell functions. TSP-1 is predominantly a negative regulator of DC and T cell function while basal SIRP-α ligation on APC by CD47 enforces tolerance. Yet, CD47/SIRP-α interaction positively controls DC and innate cell transendothelial migration. Due to the promiscuity of the protein interactions for CD47 and its ligands, it is quite interesting to note that deletion of the CD47 gene in mice largely agrees with the in vitro data with human cells. In fact, the well-conserved tissue distribution of CD47 and SIRP-α across species may facilitate the transition from bench to bedside. We thus propose CD47/TSP- 1/SIRP-α axis as an important sensor to maintain homeostasis and regulate innate and adaptive immune responses.

Background Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides. Methods and Findings In peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease. Conclusions Our work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL., In this study, Santos Susin and colleagues demonstrate that a serum-stable CD47 agonist peptide is highly effective at inducing apoptosis in chronic lymphocytic leukemia B-cells and mice., Editors’ Summary Background Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia (cancer of the white blood cells). It accounts for about a third of all adult leukemias, and about one in 200 people will develop CLL during their lifetime, usually in old age. White blood cells (including T and B lymphocytes) are made by the bone marrow (the soft center of certain bones in the body) and help the body fight infections. Leukemia begins when a bone marrow cell acquires genetic changes (mutations and chromosomal abnormalities) that make it grow and develop abnormally. Over time, the abnormal cells accumulate in the bone marrow, blood, and lymphoid organs (lymph nodes and spleen) (malignant B lymphocytes accumulate in the case of CLL) and stop the marrow producing healthy blood cells. CLL does not usually cause any symptoms during its early stages and is often diagnosed as a result of a routine blood test. Some patients, however, develop painless swellings in the lymph nodes in the neck, armpit, or groin. Other symptoms of CLL include tiredness, frequent infections, and weight loss. Treatments for CLL, which are not given unless symptoms develop, include chemotherapy, immunotherapy, and targeted therapies, which attack cancer cells without harming normal cells. Why Was This Study Done? CLL develops very slowly, and people with CLL usually live for many years after their diagnosis. However, there is no cure for CLL, and treatment fails in up to a quarter of patients because resistance to chemotherapy develops. The outlook (prognosis) is particularly bad for patients whose cancer cells contain a dysfunctional TP53 gene (TP53 encodes a protein that normally regulates cell division and cell death). Although some new therapies have recently been approved in the US and Europe, additional approaches to the treatment of CLL are still needed. One potential target for new treatments is the cell surface receptor CD47. In CLL cells, activation of CD47 by a small fragment (peptide) of thrombospondin-1 (a protein that binds to CD47) called 4N1K rapidly induces caspase-independent programmed cell death (PCD), a type of cell death in which the cell uses specialized cellular machinery to kill itself. (PCD normally controls cell numbers and eliminates cells that threaten an animal’s survival, but cancer cells often resist PCD.) Unfortunately, peptides are usually degraded quickly in the human body, which limits their usefulness as therapeutic agents. Here, therefore, the researchers investigate whether PKHB1, a variant of 4N1K designed to be stable in the body (a “serum-stable” agonist peptide), might provide a new approach to the treatment of CLL. What Did the Researchers Do and Find? The researchers tested the ability of PKHB1 to kill malignant CLL B lymphocytes grown from blood samples collected from 80 patients with CLL. PKHB1 efficiently killed these cells, including those from individuals with a dysfunctional TP53 gene but, importantly, did not kill either normal T and B lymphocytes collected from healthy donors or the residual normal T and B lymphocytes present in the patient samples. Genetic and molecular analyses undertaken by the researchers indicated that the differential response of normal and leukemic B lymphocytes to PKHB1 was the result of sustained activation of a protein called phospholipase C gamma-1 (PLCγ1) in the malignant CLL B lymphocytes. Other experiments showed that PLCγ1 activation drove the induction of a new PCD pathway (a calcium-mediated, caspase-independent pathway) that was not down-regulated by survival stimuli provided by the lymphocyte microenvironment. Finally, the researchers report that injection of PKHB1 reduced the tumor burden in a mouse model of CLL. What Do These Findings Mean? Taken together, these findings suggest that serum-stable CD47 agonist peptides represent a potential new treatment for CLL that could circumvent the problem of chemotherapy resistance and eliminate malignant CLL B lymphocytes while sparing residual normal B and T lymphocytes. These findings also demonstrate that PKHB1, the serum-stable CD47 agonist peptide tested in this study, directly induces a novel PCD pathway, thereby overcoming the innate avoidance of apoptosis by CLL cells. Before peptide-based strategies for the treatment of CLL can enter clinical trials, however, further studies are needed to develop peptides with increased stability and increased ability to bind to and activate CD47. Moreover, because the mouse CLL model used here incompletely recreates the development of CLL in people, the effectiveness and safety of this pioneer approach needs to be tested much more rigorously in animals before any clinical trials are initiated. Additional Information Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001796. The US National Cancer Institute provides information about cancer and how it develops, about targeted cancer therapies, and about CLL and its treatment (in English and Spanish) Cancer Research UK, a not-for-profit organization, provides general information about cancer and how it develops and detailed information about CLL The UK National Health Service website also provides information about CLL The American Society for Hematology provides general information about blood cancer and detailed information about leukemia, including CLL The French Society for Hematology provides general information about CLL (in French) The not-for-profit Force Hemato organization provides general information about lymphoproliferative disorders and resources for patients and families (in French) The not-for-profit organization HealthTalkOnline provides personal stories about living with CLL Wikipedia provides information about programmed cell death and about CD47 (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)

International audience; AIF is a main mediator of caspase-independent cell death. It is encoded by a single gene located on chromosome X, region q25-26 and A6 in humans and mice, respectively. Previous studies established that AIF codes for two isoforms of the protein, AIF and AIF-exB. Here, we identify a third AIF isoform resulting from an alternate transcriptional start site located at intron 9 of AIF. The resulting mRNA encodes a cytosolic protein that corresponds to the C-terminal domain of AIF (amino acids 353-613). We named this new isoform AIFshort (AIFsh). AIFsh overexpression in HeLa cells results in nuclear translocation and caspase-independent cell death. Once in the nucleus, AIFsh provokes the same effects than AIF, namely chromatin condensation and large scale (50 kb) DNA fragmentation. In contrast, these apoptogenic effects are not precluded by the AIF-inhibiting protein Hsp70. These findings identify AIFsh as a new pro-apoptotic isoform of AIF, and also reveal that the first N-terminal 352 amino acids of AIF are not required for its apoptotic activity. In addition, we demonstrate that AIFsh is strongly down-regulated in tumor cells derived from kidney, vulva, skin, thyroid, and pancreas, whereas, gamma-irradiation treatment provokes AIFsh up-regulation. Overall, our results identify a novel member of the AIF-dependent pathway and shed new light on the role of caspase-independent cell death in tumor formation/suppression.

Apoptotic cell death has been involved as a cytopathologic mechanism in response to dengue (DEN) virus infection. Little information exists about how DEN virus replication triggers apoptosis in infected cells. We reported that a nine-residue sequence of the DEN M protein referred to as ApoptoM has proapoptotic properties in transformed and tumor cells of various origins. The aim of the present study was to investigate whether ApoptoM-induced apoptosis is associated to mitochondrial dysfunction and requires caspase activation. Intracellular expression of ApoptoM provokes the disruption of the mitochondrial transmembrane potential without subsequent generation of reactive oxygen species. We showed that ApoptoM-induced apoptosis involves the activation of a caspase-like protease pathway. Caspase-3 like activity was detected in ApoptoM-expressing cells. However, there was no role for caspase-9 in ApoptoM-mediated cell death. Our data suggest that a particular mitochondrion-dependent apoptotic pathway may be involved in induction of apoptosis by ApoptoM.

The effective phagocytotic clearance of apoptotic debris is fundamental to the maintenance of neural tissues during apoptosis. Retinal photoreceptors undergo apoptosis after retinal detachment. Although their induction phase of apoptosis has been well discussed, their phagocytotic process remains quite unclear. We herein demonstrate that apoptotic photoreceptors are selectively eliminated from their physiological localization, the outer nuclear layer, to the subretinal space, and then phagocytosed by monocyte-derived macrophages. This could be shown by an ultrastructural and immunophenotypic analysis. Moreover, in chimera mice expressing transgenic green fluorescent protein in bone marrow-derived cells, the local infiltration of macrophages could be detected after retinal detachment-induced photoreceptor apoptosis. The local injection of an antibody blocking the phosphatidylserine receptor (PSR) or a peptide (GRGDSP)-blocking integrin αvβ3 revealed that phagocytotic clearance involves the PSR as well as integrin αvβ3 in vivo. Importantly, the level of blockade obtained with these reagents was different. Although anti-PSR increased the frequency of apoptotic cells that fail to bind to macrophages, GRGDSP prevented the engulfment (but not the recognition) of apoptotic photoreceptor cells by macrophages. To our knowledge, this is the first report describing the mechanisms through which apoptotic photoreceptors are selectively eliminated via a directional process in the subretinal space.

Although apoptosis is assumed to play a pivotal role in retinal function loss, its mechanism and real influence on retinal function are still unclear. To investigate the relation between retinal function and apoptosis, we studied photoreceptor apoptosis in experimental retinal detachment (RD).We induced RD by subretinal injection of sodium hyaluronate in Brown Norway rats. Apoptotic photoreceptors were detected by TdT-dUTP Terminal Nick-End Labeling (TUNEL). To evaluate the function of the detached retina, electroretinograms (ERGs) were taken on day 1, 3 with corneal electrodes and full-field stimulation.Apoptotic DNA fragmentation appeared 12 hours after RD, was most prominent on day 3, and decreased thereafter. The ERGs showed that the amplitudes of dark-adapted a-waves and light adapted 2 Hz b-waves decreased immediately after RD and continued to decrease over time. The administration of Fas/Fc chimera recombinant protein or a caspase inhibitor, Z-VAD.fmk, failed to prevent either photoreceptor apoptosis or retinal functional damage. In contrast, brain derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) significantly impeded both apoptosis and dysfunction. The ERGs recognized the functional changes sensitively, and these ERG changes correlated well to the amount of photoreceptor apoptosis. Immunohistochemical study showed that apoptosis-inducing factor (AIF), a novel caspase-independent apoptotic factor, was relocalized from mitochondria to the nucleus in this process.The present results showed that apoptosis was a key phenomenon in the retinal dysfunction in RD and that this process was transmitted mainly by mitochondria-dependent pathways rather than Fas/Fas-L or downstream caspase dependent pathways.

The apoptosis-inducing factor (AIF) is a mitochondrial-flavoprotein that, after cell death induction, is distributed to the nucleus to mediate chromatinolysis. In mitochondria, AIF is present in a monomer-dimer equilibrium that after reduction by NADH gets displaced toward the dimer. The crystal structure of the human AIF (hAIF):NAD(H)-bound dimer revealed one FAD and, unexpectedly, two NAD(H) molecules per protomer. A 1:2 hAIF:NAD(H) binding stoichiometry was additionally confirmed in solution by using surface plasmon resonance. The here newly discovered NAD(H)-binding site includes residues mutated in human disorders, and accommodation of the coenzyme in it requires restructuring of a hAIF portion within the 509-560 apoptogenic segment. Disruption of interactions at the dimerization surface by production of the hAIF E413A/R422A/R430A mutant resulted in a nondimerizable variant considerably less efficiently stabilizing charge-transfer complexes upon coenzyme reduction than WT hAIF. These data reveal that the coenzyme-mediated monomer-dimer transition of hAIF modulates the conformation of its C-terminal proapoptotic domain, as well as its mechanism as reductase. These observations suggest that both the mitochondrial and apoptotic functions of hAIF are interconnected and coenzyme controlled: a key information in the understanding of the physiological role of AIF in the cellular life and death cycle. © 2014 American Chemical Society.

Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex [1,2]. Here we show that overexpression of Hsp70 protects Apaf-1-/- cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein [3,4]. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIFDelta1-120) exhibit NADH oxidase activity, whereas superoxide anion (O(2)(-)) is formed. AIFDelta1-120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIFDelta1-120, which lacks FAD, has no NADH oxidase activity. However, native AIFDelta1-120, apoAIFDelta1-120, and the reconstituted (FAD-containing) holoAIFDelta1-120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIFDelta1-120 assessed on purified nuclei in a cell-free system. Conversely, blockade of the apoptogenic function of AIFDelta1-120 with the thiol reagent para- chloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function.

Apoptosis is nowadays what comes first to your scientist mind when someone mentions cellular suicide. However this is not the sole form of programmed cell death and many other alternative or atypical pathways have now been described. These pathways are indeed rather preferred to apoptosis in some instances based on tissue origin, cell type or development stage of the target cell. In this review, we describe many different programmed cell death subtypes according to their characteristics. Discrete, brutal, final or singular cell death pathways all participate in the elimination of unwanted, damaged or dangerous cells in organisms hence contributing to our knowledge of this particular feature of living beings: dying! Through description of anoikis, necroptosis, entosis, netosis, pyroptosis or ferroptosis, we have no choice but to realize that programmed cell death comes in many flavors.

Dolichyl monophosphate (Dol-P) has been found to induce apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5–20 min), caspase-3-like protease activation (2–4 h), chromatin condensation and DNA ladder formation (3–4 h) were observed successively. Here, we report that reduction in mitochondrial transmembrane potential and translocation of apoptosis-inducing factor (AIF) are early events (1–3 h) in the apoptotic process induced by Dol-P in U937 cells. The AIF was concentrated around nuclei and partly translocated to the nuclei, which was confirmed by immunocytochemistry using specific anti-AIF antibody. Both caspase-8 and caspase-3 inhibitors blocked only DNA fragmentation but not mitochondrial processes, AIF migration and chromatin condensation. These results indicate that mitochondrial changes are an early step in the apoptosis induced by Dol-P and AIF is one of the important factors which induce chromatin condensation in nuclei.