Targeting Brutons Tyrosine Kinase in Chronic Lymphocytic Leukemia at the Crossroad between Intrinsic and Extrinsic Pro-survival Signals

Chemo immunotherapies for chronic lymphocytic leukemia (CLL) showed a positive impact on clinical outcome, but many patients relapsed or become refractory to the available treatments. The main goal of the researchers in CLL is the identification of specific targets in order to develop new therapeutic strategies to cure the disease. The B cell receptor-signalling pathway is necessary for survival of malignant B cells and its related molecules recently become new targets for therapy. Moreover, leukemic microenvironment delivers survival signals to neoplastic cells also overcoming the apoptotic effect induced by traditional drugs. In this context, the investigation of Bruton\u2019s tyrosine kinase (Btk) is useful in: i) dissecting CLL pathogenesis; ii) finding new therapeutic approaches striking simultaneously intrinsic as well as extrinsic pro-survival signals in CLL. This paper will review these main topics

Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival

Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines.As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients

In Chronic Lymphocytic Leukemia the JAK2/STAT3 Pathway Is Constitutively Activated and Its Inhibition Leads to CLL Cell Death Unaffected by the Protective Bone Marrow Microenvironment

The bone marrow microenvironment promotes proliferation and drug resistance in chronic lymphocytic leukemia (CLL). Although ibrutinib is active in CLL, it is rarely able to clear leukemic cells protected by bone marrow mesenchymal stromal cells (BMSCs) within the marrow niche. We investigated the modulation of JAK2/STAT3 pathway in CLL by BMSCs and its targeting with AG490 (JAK2 inhibitor) or Stattic (STAT3 inhibitor). B cells collected from controls and CLL patients, were treated with medium alone, ibrutinib, JAK/Signal Transducer and Activator of Transcription (STAT) inhibitors, or both drugs, in the presence of absence of BMSCs. JAK2/STAT3 axis was evaluated by western blotting, flow cytometry, and confocal microscopy. We demonstrated that STAT3 was phosphorylated in Tyr705 in the majority of CLL patients at basal condition, and increased following co-cultures with BMSCs or IL-6. Treatment with AG490, but not Stattic, caused STAT3 and Lyn dephosphorylation, through re-activation of SHP-1, and triggered CLL apoptosis even when leukemic cells were cultured on BMSC layers. Moreover, while BMSCs hamper ibrutinib activity, the combination of ibrutinib+JAK/STAT inhibitors increase ibrutinib-mediated leukemic cell death, bypassing the pro-survival stimuli derived from BMSCs. We herein provide evidence that JAK2/STAT3 signaling might play a key role in the regulation of CLL-BMSC interactions and its inhibition enhances ibrutinib, counteracting the bone marrow niche

EVALITA Evaluation of NLP and Speech Tools for Italian - December 17th, 2020

Welcome to EVALITA 2020! EVALITA is the evaluation campaign of Natural Language Processing and Speech Tools for Italian. EVALITA is an initiative of the Italian Association for Computational Linguistics (AILC, http://www.ai-lc.it) and it is endorsed by the Italian Association for Artificial Intelligence (AIxIA, http://www.aixia.it) and the Italian Association for Speech Sciences (AISV, http://www.aisv.it)

Leukemic cell/microenvironment interactions in Chronic Lymphocytic Leukemia: role of JAK/STAT axis in the survival of neoplastic clone

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus kinases)-STAT (signal transducers and activators of transcription) pathways. During the PhD course, we particularly focused on JAK2/STAT3 axis since IL-6, one of the most abundant cytokines released in the CLL microenvironment, is the key ligand of the receptor triggering this pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. By western blotting, flow cytometry analysis, subcellular fractionation, and confocal microscopy, we analyzed 76 CLL patients and 23 healthy donors to investigate STAT3 involvement in CLL cell survival. We demonstrated that STAT3 expression was higher in malignant B cells (MFI: CLL cells 1.28±0.13 vs normals 0.61±0.09, Student’s t test, p<0.001) and the protein is significantly phosphorylated at Tyr705 compared with the normal counterpart (B lymphocytes from healthy donors) (MFI: CLL cells 211.3±35.85 vs normals 46.50±6.12, Student’s t test, p<0.05), thus showing its constitutive activation in CLL. Afterwards, we pointed out that the in vitro incubation of leukemic B cells with AG490 and Stattic, specific inhibitors of JAK2 and STAT3, respectively, induce a dose-dependent apoptosis of CLL B cells (e.g. Cell viability, 24h: CLL cells alone 62.90±4.24% vs CLL cells + AG490 50M 38.30±7.22%, Student’s t test, p<0.01; CLL cells alone 60.96±3.91% vs CLL cells + Stattic 10M 31.78±4.31%, Student’s t test, p<0.0001). Both AG490 and Stattic were able to bypass the microenvironmental protection when neoplastic B cells were co-cultured with MSCs (e.g. Cell viability, 48h: CLL cells 66.80±6.28% vs CLL cells + AG490 100M 22.50±7.50%, Student’s t test, p<0.01; CLL cells 57.51±4.95% vs CLL cells + Stattic 10M 26.25±5.45%, Student’s t test, p<0.001). In addition to JAK2/STAT3 inhibition, we showed that AG490 treatment on CLL cells can mediate other effects: i) it activates SHP-1, decreasing its phosphorylation at Ser591; ii) it inactivates protein Lyn, reducing the phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SHP-1, a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells. In conclusion, the ability of AG490 and Stattic to induce apoptosis in leukemic B cells, bypassing the pro-survival stimuli provided by the tumor microenvironment, represents a starting point for the development of new therapeutic strategies in CLL, providing at the same time new insights on the cross-talk between JAK/STAT and BCR/Lyn axes in CLL cells.La Leucemia Linfatica Cronica (LLC) è un disordine linfoproliferativo caratterizzato dall’accumulo di linfociti B maturi, con immunofenotipo CD19+/CD5+/CD23+, nel sangue periferico, in quello midollare e nei tessuti linfoidi. Nonostante le cellule leucemiche in vivo non vadano incontro ad apoptosi, se poste in vitro muoiono rapidamente; questo evidenzia l’importanza del microambiente nella sopravvivenza del clone neoplastico. Diverse molecole, incluse quelle rilasciate dalle cellule mesenchimali stromali (MSC) agiscono tramite la via di JAK (Janus Kinase)/STAT (Signal Transducer and Activators of Transcription). Nel corso del dottorato, ci siamo concentrati in particolare sull’asse JAK2/STAT3 in quanto IL-6, una delle citochine più abbondanti tra quelle presenti nel microambiente, è il ligando principale del recettore a monte di questa via. La deregolazione dell’asse JAK2/STAT3 può portare all’attivazione incontrollata di STAT3 e, conseguentemente, allo sviluppo di tumori delle cellule ematopoietiche Tramite western blotting, citometria a flusso, frazionamento subcellulare e microscopia confocale, abbiamo analizzato 76 pazienti con LLC e 23 donatori sani al fine di studiare il coinvolgimento di STAT3 nella sopravvivenza della cellula leucemica. Abbiamo dimostrato come l’espressione di STAT3 sia maggiore nelle cellule neoplastiche (MFI: cellule di LLC 1.28±0.13 vs normali 0.61±0.09, Student’s t test, p<0.001) e come sia significativamente fosforilata alla Tyr705 in confronto ai linfociti B di donatori sani (MFI: cellule di LLC 211.3±35.85 vs normali 46.50±6.12, Student’s t test, p<0.05), evidenziando così la sua attivazione costitutiva nella CLL. Dopo di ciò, abbiamo osservato come il trattamento in vitro di cellule leucemiche con AG490 e Stattic, rispettivamente inibitori specifici di JAK2 e di STAT3, induca apoptosi dose-dipendente nelle cellule B di LLC (es. Vitalità cellulare, 24h: cellule di LLC 62.90±4.24% vs cellule di LLC + AG490 50M 38.30±7.22%, Student’s t test, p<0.01; cellule di LLC 60.96±3.91% vs cellule di LLC + Stattic 10M 31.78±4.31%, Student’s t test, p<0.0001). Sia AG490 che Stattic si sono dimostrati capaci di superare la protezione fornita dal microambiente quando le cellule neoplastiche sono poste in cultura insieme alle MSC (es. Vitalità cellulare, 48h: cellule di LLC 66.80±6.28% vs cellule di LLC + AG490 100M 22.50±7.50%, Student’s t test, p<0.01; cellule di LLC 57.51±4.95% vs cellule di LLC + Stattic 10M 26.25±5.45%, Student’s t test, p<0.001). Abbiamo dimostrato come AG490 non sia solo in grado di inibire l’asse JAK2/STAT3, ma possa anche mediare altri effetti come: i) l’attivazione di SHP-1 diminuendo la sua fosforilazione in Ser591; ii) l’inibizione della proteina Lyn, riducendo la fosforilazione del sito attivo in Tyr396. Lyn, una tirosin-chinasi, e SHP-1, una tirosin-fosfatasi, sono coinvolte nella sopravvivenza delle cellule di LLC. In conclusione, la capacità di AG490 e Stattic di indurre apoptosi nelle cellule B leucemiche superando la protezione fornita dal microambiente, rappresenta un punto di partenza per lo sviluppo di nuove strategie terapeutiche nella LLC, fornendo allo stesso tempo nuove informazioni per la caratterizzazione del crosstalk tra l’asse JAK2/STAT3 e il pathway BCR/Lyn nelle cellule leucemiche

Leukemic cell/microenvironment interactions in Chronic Lymphocytic Leukemia: role of JAK/STAT axis in the survival of neoplastic clone

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus kinases)-STAT (signal transducers and activators of transcription) pathways. During the PhD course, we particularly focused on JAK2/STAT3 axis since IL-6, one of the most abundant cytokines released in the CLL microenvironment, is the key ligand of the receptor triggering this pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. By western blotting, flow cytometry analysis, subcellular fractionation, and confocal microscopy, we analyzed 76 CLL patients and 23 healthy donors to investigate STAT3 involvement in CLL cell survival. We demonstrated that STAT3 expression was higher in malignant B cells (MFI: CLL cells 1.28±0.13 vs normals 0.61±0.09, Student’s t test, p<0.001) and the protein is significantly phosphorylated at Tyr705 compared with the normal counterpart (B lymphocytes from healthy donors) (MFI: CLL cells 211.3±35.85 vs normals 46.50±6.12, Student’s t test, p<0.05), thus showing its constitutive activation in CLL. Afterwards, we pointed out that the in vitro incubation of leukemic B cells with AG490 and Stattic, specific inhibitors of JAK2 and STAT3, respectively, induce a dose-dependent apoptosis of CLL B cells (e.g. Cell viability, 24h: CLL cells alone 62.90±4.24% vs CLL cells + AG490 50M 38.30±7.22%, Student’s t test, p<0.01; CLL cells alone 60.96±3.91% vs CLL cells + Stattic 10M 31.78±4.31%, Student’s t test, p<0.0001). Both AG490 and Stattic were able to bypass the microenvironmental protection when neoplastic B cells were co-cultured with MSCs (e.g. Cell viability, 48h: CLL cells 66.80±6.28% vs CLL cells + AG490 100M 22.50±7.50%, Student’s t test, p<0.01; CLL cells 57.51±4.95% vs CLL cells + Stattic 10M 26.25±5.45%, Student’s t test, p<0.001). In addition to JAK2/STAT3 inhibition, we showed that AG490 treatment on CLL cells can mediate other effects: i) it activates SHP-1, decreasing its phosphorylation at Ser591; ii) it inactivates protein Lyn, reducing the phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SHP-1, a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells. In conclusion, the ability of AG490 and Stattic to induce apoptosis in leukemic B cells, bypassing the pro-survival stimuli provided by the tumor microenvironment, represents a starting point for the development of new therapeutic strategies in CLL, providing at the same time new insights on the cross-talk between JAK/STAT and BCR/Lyn axes in CLL cells.La Leucemia Linfatica Cronica (LLC) è un disordine linfoproliferativo caratterizzato dall’accumulo di linfociti B maturi, con immunofenotipo CD19+/CD5+/CD23+, nel sangue periferico, in quello midollare e nei tessuti linfoidi. Nonostante le cellule leucemiche in vivo non vadano incontro ad apoptosi, se poste in vitro muoiono rapidamente; questo evidenzia l’importanza del microambiente nella sopravvivenza del clone neoplastico. Diverse molecole, incluse quelle rilasciate dalle cellule mesenchimali stromali (MSC) agiscono tramite la via di JAK (Janus Kinase)/STAT (Signal Transducer and Activators of Transcription). Nel corso del dottorato, ci siamo concentrati in particolare sull’asse JAK2/STAT3 in quanto IL-6, una delle citochine più abbondanti tra quelle presenti nel microambiente, è il ligando principale del recettore a monte di questa via. La deregolazione dell’asse JAK2/STAT3 può portare all’attivazione incontrollata di STAT3 e, conseguentemente, allo sviluppo di tumori delle cellule ematopoietiche Tramite western blotting, citometria a flusso, frazionamento subcellulare e microscopia confocale, abbiamo analizzato 76 pazienti con LLC e 23 donatori sani al fine di studiare il coinvolgimento di STAT3 nella sopravvivenza della cellula leucemica. Abbiamo dimostrato come l’espressione di STAT3 sia maggiore nelle cellule neoplastiche (MFI: cellule di LLC 1.28±0.13 vs normali 0.61±0.09, Student’s t test, p<0.001) e come sia significativamente fosforilata alla Tyr705 in confronto ai linfociti B di donatori sani (MFI: cellule di LLC 211.3±35.85 vs normali 46.50±6.12, Student’s t test, p<0.05), evidenziando così la sua attivazione costitutiva nella CLL. Dopo di ciò, abbiamo osservato come il trattamento in vitro di cellule leucemiche con AG490 e Stattic, rispettivamente inibitori specifici di JAK2 e di STAT3, induca apoptosi dose-dipendente nelle cellule B di LLC (es. Vitalità cellulare, 24h: cellule di LLC 62.90±4.24% vs cellule di LLC + AG490 50M 38.30±7.22%, Student’s t test, p<0.01; cellule di LLC 60.96±3.91% vs cellule di LLC + Stattic 10M 31.78±4.31%, Student’s t test, p<0.0001). Sia AG490 che Stattic si sono dimostrati capaci di superare la protezione fornita dal microambiente quando le cellule neoplastiche sono poste in cultura insieme alle MSC (es. Vitalità cellulare, 48h: cellule di LLC 66.80±6.28% vs cellule di LLC + AG490 100M 22.50±7.50%, Student’s t test, p<0.01; cellule di LLC 57.51±4.95% vs cellule di LLC + Stattic 10M 26.25±5.45%, Student’s t test, p<0.001). Abbiamo dimostrato come AG490 non sia solo in grado di inibire l’asse JAK2/STAT3, ma possa anche mediare altri effetti come: i) l’attivazione di SHP-1 diminuendo la sua fosforilazione in Ser591; ii) l’inibizione della proteina Lyn, riducendo la fosforilazione del sito attivo in Tyr396. Lyn, una tirosin-chinasi, e SHP-1, una tirosin-fosfatasi, sono coinvolte nella sopravvivenza delle cellule di LLC. In conclusione, la capacità di AG490 e Stattic di indurre apoptosi nelle cellule B leucemiche superando la protezione fornita dal microambiente, rappresenta un punto di partenza per lo sviluppo di nuove strategie terapeutiche nella LLC, fornendo allo stesso tempo nuove informazioni per la caratterizzazione del crosstalk tra l’asse JAK2/STAT3 e il pathway BCR/Lyn nelle cellule leucemiche

Room Temperature On-Surface Synthesis of a Vinylene-Linked Single Layer Covalent Organic Framework

Conjugated single-layered two-dimensional covalent organic frameworks are flat and extended polymer networks with a unique combination of material properties, giving rise to potential applications in sensing, optoelectronics, and photonics. Despite their great potential, thus far only a few reactions to access such extended conjugated 2D polymers have been reported. Here, the on-surface polymerization of the first vinylene-linked single layered two-dimensional covalent organic framework using reversible Knoevenagel polycondensation under solvothermal conditions is described. Self-assembly of the two monomer building blocks at the solid−liquid interface led to the formation of extended covalent networks at room temperature without the need of additional catalysts or reagents. The described approach grants access to extended conjugated 2D polymers under unprecedentedly mild conditions and paves the way to new hybrid material systems

Is It Still Possible to Think about HSP70 as a Therapeutic Target in Onco-Hematological Diseases?

The search for molecules to be targeted that are involved in apoptosis resistance/increased survival and pathogenesis of onco-hematological malignancies is ongoing since these diseases are still not completely understood. Over the years, a good candidate has been identified in the Heat Shock Protein of 70kDa (HSP70), a molecule defined as “the most cytoprotective protein ever been described”. HSP70 is induced in response to a wide variety of physiological and environmental insults, allowing cells to survive lethal conditions. This molecular chaperone has been detected and studied in almost all the onco-hematological diseases and is also correlated to poor prognosis and resistance to therapy. In this review, we give an overview of the discoveries that have led us to consider HSP70 as a therapeutic target for mono- or combination-therapies in acute and chronic leukemias, multiple myeloma and different types of lymphomas. In this excursus, we will also consider HSP70 partners, such as its transcription factor HSF1 or its co-chaperones whose druggability could indirectly affect HSP70. Finally, we will try to answer the question asked in the title of this review considering that, despite the effort made by research in this field, HSP70 inhibitors never reached the clinic