Clinical and biological features of B-cell neoplasms with CDK6 translocations: an association with a subgroup of splenic marginal zone lymphomas displaying frequent CD5 expression, prolymphocytic cells, and TP53 abnormalities.

A translocation involving the cyclin-dependent kinase 6 (CDK6) gene [t(CDK6)] is a rare but recurrent abnormality in B-cell neoplasms. To further characterise this aberration, we studied 57 cases; the largest series reported to date. Fluorescence in situ hybridisation analysis confirmed the involvement of CDK6 in all cases, including t(2;7)(p11;q21) immunoglobulin kappa locus (IGK)/CDK6 (n = 51), t(7;14)(q21;q32) CDK6/immunoglobulin heavy locus (IGH) (n = 2) and the previously undescribed t(7;14)(q21;q11) CDK6/T-cell receptor alpha locus (TRA)/T-cell receptor delta locus (TRD) (n = 4). In total, 10 patients were diagnosed with chronic lymphocytic leukaemia, monoclonal B-cell lymphocytosis or small lymphocytic lymphoma, and 47 had small B-cell lymphoma (SmBL) including 36 cases of marginal zone lymphoma (MZL; 34 splenic MZLs, one nodal MZL and one bronchus-associated lymphoid tissue lymphoma). In all, 18 of the 26 cytologically reviewed cases of MZL (69%) had an atypical aspect with prolymphocytic cells. Among the 47 patients with MZL/SmBL, CD5 expression was found in 26 (55%) and the tumour protein p53 (TP53) deletion in 22 (47%). The TP53 gene was mutated in 10/30 (33%); the 7q deletion was detected in only one case, and no Notch receptor 2 (NOTCH2) mutations were found. Immunoglobulin heavy-chain variable-region (IGHV) locus sequencing revealed that none harboured an IGHV1-02*04 gene. Overall survival was 82% at 10 years and not influenced by TP53 aberration. Our present findings suggest that most t(CDK6)+ neoplasms correspond to a particular subgroup of indolent marginal zone B-cell lymphomas with distinctive features.

Genetic characterization of B-cell prolymphocytic leukemia: a prognostic model involving MYC and TP53.

B-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessments of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex (≥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 (76%) of the 34 patients exhibited an 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 3 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 inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We concluded that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease.

CD47 agonist peptide PKHB1 induces immunogenic cell death in T-cell acute lymphoblastic leukemia cells.

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.

"Double-hit" chronic lymphocytic leukemia: An aggressive subgroup with 17p deletion and 8q24 gain.

Chronic lymphocytic leukemia (CLL) with 17p deletion (17p-) is associated with a lack of response to standard treatment and thus the worst possible clinical outcome. Various chromosomal abnormalities (including unbalanced translocations, deletions, ring chromosomes and isochromosomes) result in the loss of 17p and one copy of the TP53 gene. The objective of the present study was to determine whether the type of chromosomal abnormality leading to 17p- and the additional aberrations influenced the prognosis in a series of 195 patients with 17p-CLL. Loss of 17p resulted primarily from an unbalanced translocation (70%) with several chromosome partners (the most frequent being chromosome 18q), followed by deletion 17p (23%), monosomy 17 (8%), isochromosome 17q [i(17q)] (5%) and a ring chromosome 17 (2%). In a univariate analysis, monosomy 17, a highly complex karyotype (≥5 abnormalities), and 8q24 gain were associated with poor treatment-free survival, and i(17q) (P = .04), unbalanced translocations (P = .03) and 8q24 gain (P = .001) were significantly associated with poor overall survival. In a multivariate analysis, 8q24 gain remained a significant predictor of poor overall survival. We conclude that 17p deletion and 8q24 gain have a synergistic impact on outcome, and so patients with this "double-hit" CLL have a particularly poor prognosis. Systematic, targeting screening for 8q24 gain should therefore be considered in cases of 17p- CLL.

Key Residues Regulating the Reductase Activity of the Human Mitochondrial Apoptosis Inducing Factor.

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.

CD47 agonist peptides induce programmed cell death in refractory chronic lymphocytic leukemia B cells via PLCγ1 activation: evidence from mice and humans.

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.

Structural insights into the coenzyme mediated monomer-dimer transition of the pro-apoptotic apoptosis inducing factor.

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.

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX.

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

CD47(high) expression on CD4 effectors identifies functional long-lived memory T cell progenitors.

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 CD47(high) to CD47(low) to CD47(high), during the course of the in vivo IR. Specifically, CD47(high) 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 CD47(high), but not CD47(low), polyclonal effector CD4 T cells during recall responses in an experimental model of chronic airway inflammatory disease. Passive transfer of CD47(high), but not CD47(low), CD4 T cells in nonlymphopenic naive mice generated long-lived memory T cells capable of anamnestic responses. We conclude that CD47(high) status on CD4 T cells identifies functional long-lived memory T cell progenitors.

CD38/NAD+ glycohydrolase and associated antigens in chronic lymphocytic leukaemia: From interconnected signalling pathways to therapeutic strategies.

Chronic lymphocytic leukaemia (CLL) is a heterogenous disease characterized by the accumulation of neoplastic CD5+/CD19+ B lymphocytes. The spreading of the leukaemia relies on the CLL cell's ability to survive in the blood and migrate to and proliferate within the bone marrow and lymphoid tissues. Some patients with CLL are either refractory to the currently available therapies or relapse after treatment; this emphasizes the need for novel therapeutic strategies that improving clinical responses and overcome drug resistance. CD38 is a marker of a poor prognosis and governs a set of survival, proliferation and migration signals that contribute to the pathophysiology of CLL. The literature data evidence a spatiotemporal association between the cell surface expression of CD38 and that of other CLL antigens, such as the B-cell receptor (BCR), CD19, CD26, CD44, the integrin very late antigen 4 (VLA4), the chemokine receptor CXCR4, the vascular endothelial growth factor receptor-2 (VEGF-R2), and the neutrophil gelatinase-associated lipocalin receptor (NGAL-R). Most of these proteins contribute to CLL cell survival, proliferation and trafficking, and cooperate with CD38 in multilayered signal transduction processes. In general, these antigens have already been validated as therapeutic targets in cancer, and a broad repertoire of specific monoclonal antibodies and derivatives are available. Here, we review the state of the art in this field and examine the therapeutic opportunities for cotargeting CD38 and its partners in CLL, e.g. by designing novel bi-/trispecific antibodies.

ACOX1-mediated peroxisomal fatty acid oxidation contributes to metabolic reprogramming and survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is still an incurable disease, with many patients developing resistance to conventional and targeted therapies. To better understand the physiology of CLL and facilitate the development of innovative treatment options, we examined specific metabolic features in the tumor CLL B-lymphocytes. We observed metabolic reprogramming, characterized by a high level of mitochondrial oxidative phosphorylation activity, a low glycolytic rate, and the presence of C2- to C6-carnitine end-products revealing an unexpected, essential role for peroxisomal fatty acid beta-oxidation (pFAO). Accordingly, downmodulation of ACOX1 (a rate-limiting pFAO enzyme overexpressed in CLL cells) was enough to shift the CLL cells' metabolism from lipids to a carbon- and amino-acid-based phenotype. Complete blockade of ACOX1 resulted in lipid droplet accumulation and caspase-dependent death in CLL cells, including those from individuals with poor cytogenetic and clinical prognostic factors. In a therapeutic translational approach, ACOX1 inhibition spared non-tumor blood cells from CLL patients but led to the death of circulating, BCR-stimulated CLL B-lymphocytes and CLL B-cells receiving pro-survival stromal signals. Furthermore, a combination of ACOX1 and BTK inhibitors had a synergistic killing effect. Overall, our results highlight a less-studied but essential metabolic pathway in CLL and pave the way towards the development of new, metabolism-based treatment options.

[Programmed cell death comes in many flavors]. / La mort cellulaire programmée ne manque pas de vocabulaire.

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.

Extracellular Vesicles in Chronic Lymphocytic Leukemia: Tumor Microenvironment Messengers as a Basis for New Targeted Therapies?

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).

The Value of Neutrophil Gelatinase-Associated Lipocalin Receptor as a Novel Partner of CD38 in Chronic Lymphocytic Leukemia: From an Adverse Prognostic Factor to a Potential Pharmacological Target?

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of neoplastic B lymphocytes that escape death, and correlates with the expression of negative prognostic markers such as the CD38 antigen. Although certain new drugs approved by the US Food and Drug Administration improve the clinical outcome of CLL patients, drug resistance and disease relapse still occur. Like CD38, neutrophil gelatinase-associated lipocalin receptor (NGAL-R) is frequently overexpressed in CLL cells. Here, we evaluated the concomitant surface expression of NGAL-R and CD38 in leukemic blood cells from 52 CLL patients (37 untreated, 8 in clinical remission, and 7 relapsed). We provide evidence of a positive correlation between NGAL-R and CD38 levels both in the interpatient cohorts (p < 0.0001) and in individual patients, indicating a constitutive association of NGAL-R and CD38 at the cell level. Patients with progressing CLL showed a time-dependent increase in NGAL-R/CD38 levels. In treated CLL patients who achieved clinical remission, NGAL-R/CD38 levels were decreased, and were significantly lower than in the untreated and relapsed groups (p < 0.02). As NGAL-R and CD38 participate in CLL cell survival, envisioning their simultaneous inhibition with bispecific NGAL-R/CD38 antibodies capable of inducing leukemic cell death might provide therapeutic benefit for CLL patients.

del(8p) and TNFRSF10B loss are associated with a poor prognosis and resistance to fludarabine in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, the prognosis of which varies according to the cytogenetic group. We characterized a rare chromosomal abnormality (del(8p), deletion of the short arm of chromosome 8) in the context of CLL. By comparing the largest cohort of del(8p) CLL to date (n = 57) with a non-del(8p) cohort (n = 155), del(8p) was significantly associated with a poor prognosis, a shorter time to first treatment, worse overall survival (OS), and a higher risk of Richter transformation. For patients treated with fludarabine-based regimens, the next-treatment-free survival and the OS were shorter in del(8p) cases (including those with mutated IGHV). One copy of the TNFRSF10B gene (coding a pro-apoptotic receptor activated by TRAIL) was lost in 91% of del(8p) CLL. TNFRSF10B was haploinsufficient in del(8p) CLL, and was involved in the modulation of fludarabine-induced cell death - as confirmed by our experiments in primary cells and in CRISPR-edited TNFRSF10B knock-out CLL cell lines. Lastly, del(8p) abrogated the synergy between fludarabine and TRAIL-induced apoptosis. Our results highlight del(8p)'s value as a prognostic marker and suggest that fit CLL patients (i.e. with mutated IGHV and no TP53 disruption) should be screened for del(8p) before the initiation of fludarabine-based treatment.

Current Status of Novel Agents for the Treatment of B Cell Malignancies: What's Coming Next?

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.

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network.

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

AIF-mediated caspase-independent necroptosis: a new chance for targeted therapeutics.

Cell death has been initially divided into apoptosis, in which the cell plays an active role, and necrosis, which is considered a passive cell death program. Intense research performed in the last decades has concluded that "programmed" cell death (PCD) is a more complex physiological process than initially thought. Indeed, although in most cases the PCD process is achieved via a family of Cys proteases known as caspases, an important number of regulated PCD pathways do not involve this family of proteases. As a consequence, active forms of PCD are initially referred to as caspase-dependent and caspase-independent. More recent data has revealed that there are also active caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). The existence of necroptotic forms of death was corroborated by the discovery of key executioners such as the kinase RIP1 or the mitochondrial protein apoptosis-inducing factor (AIF). AIF is a Janus protein with a redox activity in the mitochondria and a pro-apoptotic function in the nucleus. We have recently described a particular form of AIF-mediated caspase-independent necroptosis that also implicates other molecules such as PARP-1, calpains, Bax, Bcl-2, histone H2AX, and cyclophilin A. From a therapeutic point of view, the unraveling of this new form of PCD poses a question: is it possible to modulate this necroptotic pathway independently of the classical apoptotic paths? Because the answer is yes, a wider understanding of AIF-mediated necroptosis could theoretically pave the way for the development of new drugs that modulate PCD. To this end, we present here an overview of the current knowledge of AIF and AIF-mediated necroptosis. We also summarize the state of the art in some of the most interesting therapeutic strategies that could target AIF or the AIF-mediated necroptotic pathway.

Activation of Interferon Signaling in Chronic Lymphocytic Leukemia Cells Contributes to Apoptosis Resistance via a JAK-Src/STAT3/Mcl-1 Signaling Pathway.

Besides their antiviral and immunomodulatory functions, type I (α/ß) and II (γ) interferons (IFNs) exhibit either beneficial or detrimental effects on tumor progression. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of abnormal CD5+ B lymphocytes that escape death. Drug resistance and disease relapse still occur in CLL. The triggering of IFN receptors is believed to be involved in the survival of CLL cells, but the underlying molecular mechanisms are not yet characterized. We show here that both type I and II IFNs promote the survival of primary CLL cells by counteracting the mitochondrial (intrinsic) apoptosis pathway. The survival process was associated with the upregulation of signal transducer and activator of transcription-3 (STAT3) and its target anti-apoptotic Mcl-1. Furthermore, the blockade of the STAT3/Mcl-1 pathway by pharmacological inhibitors against STAT3, TYK2 (for type I IFN) or JAK2 (for type II IFN) markedly reduced IFN-mediated CLL cell survival. Similarly, the selective Src family kinase inhibitor PP2 notably blocked IFN-mediated CLL cell survival by downregulating the protein levels of STAT3 and Mcl-1. Our work reveals a novel mechanism of resistance to apoptosis promoted by IFNs in CLL cells, whereby JAKs (TYK2, JAK2) and Src kinases activate in concert a STAT3/Mcl-1 signaling pathway. In view of current clinical developments of potent STAT3 and Mcl-1 inhibitors, a combination of conventional treatments with these inhibitors might thus constitute a new therapeutic strategy in CLL.

Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis.

Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N'-nitro-N'-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.