Decreased apoptotic priming and loss of BCL-2 dependence are functional hallmarks of Richter's syndrome.

Richter's syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.

In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells.

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF.

Theratyping of the Rare CFTR Genotype A559T in Rectal Organoids and Nasal Cells Reveals a Relevant Response to Elexacaftor (VX-445) and Tezacaftor (VX-661) Combination.

Despite the promising results of new CFTR targeting drugs designed for the recovery of F508del- and class III variants activity, none of them have been approved for individuals with selected rare mutations, because uncharacterized CFTR variants lack information associated with the ability of these compounds in recovering their molecular defects. Here we used both rectal organoids (colonoids) and primary nasal brushed cells (hNEC) derived from a CF patient homozygous for A559T (c.1675G>A) variant to evaluate the responsiveness of this pathogenic variant to available CFTR targeted drugs that include VX-770, VX-809, VX-661 and VX-661 combined with VX-445. A559T is a rare mutation, found in African-Americans people with CF (PwCF) with only 85 patients registered in the CFTR2 database. At present, there is no treatment approved by FDA (U.S. Food and Drug Administration) for this genotype. Short-circuit current (Isc) measurements indicate that A559T-CFTR presents a minimal function. The acute addition of VX-770 following CFTR activation by forskolin had no significant increment of baseline level of anion transport in both colonoids and nasal cells. However, the combined treatment, VX-661-VX-445, significantly increases the chloride secretion in A559T-colonoids monolayers and hNEC, reaching approximately 10% of WT-CFTR function. These results were confirmed by forskolin-induced swelling assay and by western blotting in rectal organoids. Overall, our data show a relevant response to VX-661-VX-445 in rectal organoids and hNEC with CFTR genotype A559T/A559T. This could provide a strong rationale for treating patients carrying this variant with VX-661-VX-445-VX-770 combination.

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking.

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.

Effects of Magnetic Nanoparticles on the Functional Activity of Human Monocytes and Dendritic Cells.

The use of nanoparticles in medicine is sometimes hampered by their potential to activate immune cells, eliciting inflammation or allergy. We investigated whether magnetic nanoparticles (MNPs) or biomimetic magnetic nanoparticles (BMNPs) affect relevant activities of human monocytes. We found that the nanoparticles neither elicited the production of pro-inflammatory mediators IL-6 and TNFα by resting monocytes (when BMNP dose < 300 µg/mL) nor enhanced their secretion induced by R848, a molecule engaging virus-recognizing receptors, or bacterial lipopolysaccharide (LPS). MNPs and BMNPs neither induced the generation of reactive oxygen species (ROS), nor affected the ROS production elicited by the NADPH oxidase activator phorbol myristate acetate (PMA) or the fungal derivative ß-glucan. BMNPs, but not MNPs, caused an up-regulation of the maturation markers CD80, CD83, and CD86 in immature monocyte-derived dendritic cells (DCs), whereas both nanoparticles did not affect the LPS-induced expression of these markers. Moreover, the nanoparticles were greedily ingested by monocytes and DCs without altering their viability. Therefore, these nanoparticles are candidates for medical applications because they do not activate pro-inflammatory activities of monocytes. Furthermore, their ability to stimulate DC maturation could be used for the design of vaccines. Moreover, harmlessly engulfed nanoparticles could be vehicles to carry molecules inside the immune cells to regulate the immune response.

Atypical CXCL12 signaling enhances neutrophil migration by modulating nuclear deformability.

To reach inflamed tissues from the circulation, neutrophils must overcome physical constraints imposed by the tissue architecture, such as the endothelial barrier or the three-dimensional (3D) interstitial space. In these microenvironments, neutrophils are forced to migrate through spaces smaller than their own diameter. One of the main challenges for cell passage through narrow gaps is the deformation of the nucleus, the largest and stiffest organelle in cells. Here, we showed that chemokines, the extracellular signals that guide cell migration in vivo, modulated nuclear plasticity to support neutrophil migration in restricted microenvironments. Exploiting microfabricated devices, we found that the CXC chemokine CXCL12 enhanced the nuclear pliability of mouse bone marrow-derived neutrophils to sustain their migration in 3D landscapes. This previously uncharacterized function of CXCL12 was mediated by the atypical chemokine receptor ACKR3 (also known as CXCR7), required protein kinase A (PKA) activity, and induced chromatin compaction, which resulted in enhanced cell migration in 3D. Thus, we propose that chemical cues regulate the nuclear plasticity of migrating leukocytes to optimize their motility in restricted microenvironments.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

A Comprehensive Review of Receptor-Type Tyrosine-Protein Phosphatase Gamma (PTPRG) Role in Health and Non-Neoplastic Disease.

Protein tyrosine phosphatase receptor gamma (PTPRG) is known to interact with and regulate several tyrosine kinases, exerting a tumor suppressor role in several type of cancers. Its wide expression in human tissues compared to the other component of group 5 of receptor phosphatases, PTPRZ expressed as a chondroitin sulfate proteoglycan in the central nervous system, has raised interest in its role as a possible regulatory switch of cell signaling processes. Indeed, a carbonic anhydrase-like domain (CAH) and a fibronectin type III domain are present in the N-terminal portion and were found to be associated with its role as [HCO3-] sensor in vascular and renal tissues and a possible interaction domain for cell adhesion, respectively. Studies on PTPRG ligands revealed the contactins family (CNTN) as possible interactors. Furthermore, the correlation of PTPRG phosphatase with inflammatory processes in different normal tissues, including cancer, and the increasing amount of its soluble form (sPTPRG) in plasma, suggest a possible role as inflammatory marker. PTPRG has important roles in human diseases; for example, neuropsychiatric and behavioral disorders and various types of cancer such as colon, ovary, lung, breast, central nervous system, and inflammatory disorders. In this review, we sum up our knowledge regarding the latest discoveries in order to appreciate PTPRG function in the various tissues and diseases, along with an interactome map of its relationship with a group of validated molecular interactors.

CCR7 signalosomes are preassembled on tips of lymphocyte microvilli in proximity to LFA-1.

Leukocyte microvilli are elastic actin-rich projections implicated in rapid sensing and penetration across glycocalyx barriers. Microvilli are critical for the capture and arrest of flowing lymphocytes by high endothelial venules, the main lymph node portal vessels. T lymphocyte arrest involves subsecond activation of the integrin LFA-1 by the G-protein-coupled receptor CCR7 and its endothelial-displayed ligands, the chemokines CCL21 and CCL19. The topographical distribution of CCR7 and of LFA-1 in relation to lymphocyte microvilli has never been elucidated. We applied the recently developed microvillar cartography imaging technique to determine the topographical distribution of CCR7 and LFA-1 with respect to microvilli on peripheral blood T lymphocytes. We found that CCR7 is clustered on the tips of T cell microvilli. The vast majority of LFA-1 molecules were found on the cell body, likely assembled in macroclusters, but a subset of LFA-1, 5% of the total, were found scattered within 20 nm from the CCR7 clusters, implicating these LFA-1 molecules as targets for inside-out activation signals transmitted within a fraction of a second by chemokine-bound CCR7. Indeed, RhoA, the key GTPase involved in rapid LFA-1 affinity triggering by CCR7, was also found to be clustered near CCR7. In addition, we observed that the tyrosine kinase JAK2 controls CCR7-mediated LFA-1 affinity triggering and is also highly enriched on tips of microvilli. We propose that tips of lymphocyte microvilli are novel signalosomes for subsecond CCR7-mediated inside-out signaling to neighboring LFA-1 molecules, a critical checkpoint in LFA-1-mediated lymphocyte arrest on high endothelial venules.

Oxyresveratrol-Loaded PLGA Nanoparticles Inhibit Oxygen Free Radical Production by Human Monocytes: Role in Nanoparticle Biocompatibility.

Oxyresveratrol, a polyphenol extracted from the plant Artocarpus lakoocha Roxb, has been reported to be an antioxidant and an oxygen-free radical scavenger. We investigated whether oxyresveratrol affects the generation of superoxide anion (O2-) by human monocytes, which are powerful reactive oxygen species (ROS) producers. We found that oxyresveratrol inhibited the O2- production induced upon stimulation of monocytes with ß-glucan, a well known fungal immune cell activator. We then investigated whether the inclusion of oxyresveratrol into nanoparticles could modulate its effects on O2- release. We synthesized poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and we assessed their effects on monocytes. We found that empty PLGA nanoparticles induced O2- production by resting monocytes and enhanced the formation of this radical in ß-glucan-stimulated monocytes. Interestingly, the insertion of oxyresveratrol into PLGA nanoparticles significantly inhibited the O2- production elicited by unloaded nanoparticles in resting monocytes as well as the synergistic effect of nanoparticles and ß-glucan. Our results indicate that oxyresveratrol is able to inhibit ROS production by activated monocytes, and its inclusion into PLGA nanoparticles mitigates the oxidative effects due to the interaction between these nanoparticles and resting monocytes. Moreover, oxyresveratrol can contrast the synergistic effects of nanoparticles with fungal agents that could be present in the patient tissues. Therefore, oxyresveratrol is a natural compound able to make PLGA nanoparticles more biocompatible.

Analysing omics data sets with weighted nodes networks (WNNets).

Current trends in biomedical research indicate data integration as a fundamental step towards precision medicine. In this context, network models allow representing and analysing complex biological processes. However, although effective in unveiling network properties, these models fail in considering the individual, biochemical variations occurring at molecular level. As a consequence, the analysis of these models partially loses its predictive power. To overcome these limitations, Weighted Nodes Networks (WNNets) were developed. WNNets allow to easily and effectively weigh nodes using experimental information from multiple conditions. In this study, the characteristics of WNNets were described and a proteomics data set was modelled and analysed. Results suggested that degree, an established centrality index, may offer a novel perspective about the functional role of nodes in WNNets. Indeed, degree allowed retrieving significant differences between experimental conditions, highlighting relevant proteins, and provided a novel interpretation for degree itself, opening new perspectives in experimental data modelling and analysis. Overall, WNNets may be used to model any high-throughput experimental data set requiring weighted nodes. Finally, improving the power of the analysis by using centralities such as betweenness may provide further biological insights and unveil novel, interesting characteristics of WNNets.

Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells.

The regulatory role of protein tyrosine kinases in ß1- and ß2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

Synergistic efficacy of the dual PI3K-δ/γ inhibitor duvelisib with the Bcl-2 inhibitor venetoclax in Richter syndrome PDX models.

A small subset of cases of chronic lymphocytic leukemia undergoes transformation to diffuse large B-cell lymphoma, Richter syndrome (RS), which is associated with a poor prognosis. Conventional chemotherapy results in limited responses, underlining the need for novel therapeutic strategies. Here, we investigate the ex vivo and in vivo efficacy of the dual phosphatidylinositol 3-kinase-δ/γ (PI3K-δ/γ) inhibitor duvelisib (Duv) and the Bcl-2 inhibitor venetoclax (Ven) using 4 different RS patient-derived xenograft (PDX) models. Ex vivo exposure of RS cells to Duv, Ven, or their combination results in variable apoptotic responses, in line with the expression levels of target proteins. Although RS1316, IP867/17, and RS9737 cells express PI3K-δ, PI3K-γ, and Bcl-2 and respond to the drugs, RS1050 cells, expressing very low levels of PI3K-γ and lacking Bcl-2, are fully resistant. Moreover, the combination of these drugs is more effective than each agent alone. When tested in vivo, RS1316 and IP867/17 show the best tumor growth inhibition responses, with the Duv/Ven combination leading to complete remission at the end of treatment. The synergistic effect of Duv and Ven relies on the crosstalk between PI3K and apoptotic pathways occurring at the GSK3ß level. Indeed, inhibition of PI3K signaling by Duv results in GSK3ß activation, leading to ubiquitination and subsequent degradation of both c-Myc and Mcl-1, making RS cells more sensitive to Bcl-2 inhibition by Ven. This work provides, for the first time, a proof of concept of the efficacy of dual targeting of PI3K-δ/γ and Bcl-2 in RS and providing an opening for a Duv/Ven combination for these patients. Clinical studies in aggressive lymphomas, including RS, are under way. This trial was registered at www.clinicaltrials.gov as #NCT03892044.

Monocyte-to-macrophage switch reversibly impaired by Ibrutinib.

Ibrutinib is increasingly adopted for treating lymphoid malignancies. While growing amounts of data pile up about Ibrutinib mechanism of action on neoplastic B cells, little is known about its impact on other immune cells. Here we investigated the effect of Ibrutinib on monocyte/macrophage functions. (1) Ibrutinib treatment of purified human monocytes affected both chemoattractant-triggered inside-out as well as integrin-mediated outside-in signaling events, thus provoking defective adhesion and spreading on purified integrin ligands, respectively. (2) In in vitro cell-culture experiments, Ibrutinib promoted a differentiation shift of monocytes to fibrocyte-like cells, characterized by the acquisition of a typical elongated cell morphology. Importantly, this clear-cut shape transition also occurred upon culturing monocytes with sera derived from Ibrutinib-treated patients, thus clearly suggesting that the drug concentrations achievable in vivo can generate the phenotypic shift. (3) Ibrutinib-induced fibrocyte-like cells showed adhesion deficiency, altered phagocytic properties, and, with respect to macrophages, they acquired the capability of generating larger amounts of reactive oxygen species, possibly displaying different metabolic activities. Taken together, our results indicate that Ibrutinib has profound effects on the monocyte/macrophage immunobiology. They may finally shed some light about the biological ground of several Ibrutinib-related toxicities.

CXCR4- and BCR-triggered integrin activation in B-cell chronic lymphocytic leukemia cells depends on JAK2-activated Bruton's tyrosine kinase.

Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment.

Efficient lysis of B-chronic lymphocytic leukemia cells by the plant-derived sesquiterpene alcohol α-bisabolol, a dual proapoptotic and antiautophagic agent.

The sesquiterpene α-bisabolol (α-BSB) is a cytotoxic agent against acute leukemia and chronic myeloid leukemia cells. Here the profile of α-BSB citotoxicity was evaluated ex vivo in primary mononuclear blood cells isolated from 45 untreated B-chronic lymphocytic leukemia (B-CLL) patients. We studied the effects of α-BSB by flow cytometric and western blotting techniques with the following findings: (1) α-BSB was an effective proapoptotic agent against B-CLL cells (IC50 42 ± 15 µM). It was also active, but to a lesser extent, on normal residual B cells and monocytes (IC50 68 ± 34 and 74 ± 28 µM, respectively; p < 0.01), while T-cells, though not achieving IC50, were nevertheless decreased. (2) Lipid raft content positively correlated with α-BSB cell sensitivity, while neither the phenotype of B-CLL cells nor the disease clinical stage did affect the sensitivity to α-BSB. (3) Flow cytometry analysis evidenced the induction of pores in mitochondrial and lysosomal membrane after 3- to 5-hour exposure of B-CLL cells to α-BSB, leading to apoptosis; in contrast, western blotting analysis showed inhibition of the autophagic flux. Therefore, according to cellular selectivity, α-BSB is a cytotoxic agent preferentially active against leukemic cells, while its lower activity on normal B cells, monocytes and T cells may account for an additive anti-inflammatory effect targeting the leukemia-associated pro-inflammatory microenvironment. Consistent with the observed effects on intracellular processes, α-BSB should be regarded as a dual agent, both activating mitochondrial-based apoptosis and inhibiting autophagy by disrupting lysosomes.

Low catalase expression confers redox hypersensitivity and identifies an indolent clinical behavior in CLL.

B-cell receptor (BCR) signaling is a key determinant of variable clinical behavior and a target for therapeutic interventions in chronic lymphocytic leukemia (CLL). Endogenously produced H2O2 is thought to fine-tune the BCR signaling by reversibly inhibiting phosphatases. However, little is known about how CLL cells sense and respond to such redox cues and what effect they have on CLL. We characterized the response of BCR signaling proteins to exogenous H2O2 in cells from patients with CLL, using phosphospecific flow cytometry. Exogenous H2O2 in the absence of BCR engagement induced a signaling response of BCR proteins that was higher in CLL with favorable prognostic parameters and an indolent clinical course. We identified low catalase expression as a possible mechanism accounting for redox signaling hypersensitivity. Decreased catalase could cause an escalated accumulation of exogenous H2O2 in leukemic cells with a consequent greater inhibition of phosphatases and an increase of redox signaling sensitivity. Moreover, lower levels of catalase were significantly associated with a slower progression of the disease. In leukemic cells characterized by redox hypersensitivity, we also documented an elevated accumulation of ROS and an increased mitochondrial amount. Taken together, our data identified redox sensitivity and metabolic profiles that are linked to differential clinical behavior in CLL. This study advances our understanding of the redox and signaling heterogeneity of CLL and provides the rationale for the development of therapies targeting redox pathways in CLL.

A single amino acid substitution confers B-cell clonogenic activity to the HIV-1 matrix protein p17.

Recent data highlight the presence, in HIV-1-seropositive patients with lymphoma, of p17 variants (vp17s) endowed with B-cell clonogenicity, suggesting a role of vp17s in lymphomagenesis. We investigated the mechanisms responsible for the functional disparity on B cells between a wild-type p17 (refp17) and a vp17 named S75X. Here, we show that a single Arginine (R) to Glycine (G) mutation at position 76 in the refp17 backbone (p17R76G), as in the S75X variant, is per se sufficient to confer a B-cell clonogenic potential to the viral protein and modulate, through activation of the PTEN/PI3K/Akt signaling pathway, different molecules involved in apoptosis inhibition (CASP-9, CASP-7, DFF-45, NPM, YWHAZ, Src, PAX2, MAPK8), cell cycle promotion and cancer progression (CDK1, CDK2, CDK8, CHEK1, CHEK2, GSK-3 beta, NPM, PAK1, PP2C-alpha). Moreover, the only R to G mutation at position 76 was found to strongly impact on protein folding and oligomerization by altering the hydrogen bond network. This generates a conformational shift in the p17 R76G mutant which enables a functional epitope(s), masked in refp17, to elicit B-cell growth-promoting signals after its interaction with a still unknown receptor(s). Our findings offer new opportunities to understand the molecular mechanisms accounting for the B-cell growth-promoting activity of vp17s.

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage.

CCRL2 is a 7-transmembrane domain receptor that shares structural and functional similarities with the family of atypical chemokine receptors (ACKRs). CCRL2 is upregulated by inflammatory signals and, unlike other ACKRs, it is not a chemoattractant-scavenging receptor, does not activate ß-arrestins, and is widely expressed by many leukocyte subsets. Therefore, the biological role of CCRL2 in immunity is still unclear. We report that CCRL2-deficient mice have a defect in neutrophil recruitment and are protected in 2 models of inflammatory arthritis. In vitro, CCRL2 was found to constitutively form homodimers and heterodimers with CXCR2, a main neutrophil chemotactic receptor. By heterodimerization, CCRL2 could regulate membrane expression and promote CXCR2 functions, including the activation of ß2-integrins. Therefore, upregulation of CCRL2 observed under inflammatory conditions is functional to finely tune CXCR2-mediated neutrophil recruitment at sites of inflammation.