Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

VSV Oncolysis in Combination With the BCL-2 Inhibitor Obatoclax Overcomes Apoptosis Resistance in Chronic Lymphocytic Leukemia

Clinical utility of assessing ZAP‐70 and CD38 in chronic lymphocytic leukemia

Novel Oncogenic Non-Coding RNA：circRIC8B Regulates Lipid Metabolism Via Mir-199b-5p /LPL Axis in Chronic Lymphocytic Leukemia

Background. Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease that manifests by the accumulation of tumor monoclonal B-lymphocytes in the bone marrow, peripheral blood and secondary lymphoid organs. Recently it was found that CLL cells are able to form immunological synapses with microenvironment cells, directly and indirectly affecting their function. Therefore, it became clear that the pathogenesis of CLL is not only escape of apoptosis but also the ability of CLL cells to cause T-lymphocyte anergy, thereby avoiding immune surveillance. Objective: to study the expression of FAS, co-stimulatory molecules CD80 and CD86, PD-1, PD-L1 on CLL cells, and also to study the basic subpopulations of T-cells (naїve, memory, effector cells). Materials and methods. The study included 46 CLL patients: 16 patients with disease progression after chemotherapy and 30 patients with newly diagnosed CLL. “Primary” patients are categorized according to J. Binet’s CLL stages. Stage A was established in 14 patients, B – 10, C – 6. The control group included 29 healthy donors. Peripheral blood was used as a material for analysis. The study was performed on a 6-color flow cytometer BD FACS Canto II (BD Biosciences, USA). Results. In CLL patients, the proportion of CD80+, CD86+, FAS+ B-cells was significantly lower than in donors. In “primary” patients the proportion of CD80 + CLL cells was higher than in patients with CLL progression. Among “primary” patients the proportion of CD80+ and CD86+ was lower in advanced stages of the disease. In patients with CLL progression the proportion of FAS+ B cells was higher than in “primary” patients. The proportion of PD-1+ B cells in CLL patients was higher than in donors and “primary” patients. The proportion of PD-1+ tumor cells was significantly lower in advanced stages of the disease. The proportion of PD-L1+ B cells in CLL patients was lower than in donors. Among the “primary” patients, the proportion of PD-L1+ B-lymphocytes was higher in stage A. The proportion of PD-1+ Thelpers was higher in CLL patients than in donors, and among “primary” patients it was higher in advanced stages of the disease. The proportion of PDL1+ T-helpers and cytotoxic T-cells in CLL patients was lower than in donors. The proportion of naїve cells (CD95-CD28+) in patients compared with donors was lower and the proportion of effector cells (CD95+ CD28–), memory cells (CD95 + CD28 +) was higher, a proportion of CD8+ memory T-cells was higher among patients in the advanced stage of CLL. Conclusion . Therefore, a decline the CD80/CD86+ B-cells in CLL can cause ineffectiveness of an immunological synapse between tumor cells and T-cells, which leads to anergy of T-lymphocytes. Decline expression of the FAS receptor allows tumor CLL cells to avoid FAS-mediated apoptosis. A change in the T-cell pool toward memory cells and effectors, the acquisition of a CD4+PD-1+ (“exhausted”) phenotype impaired antitumor immunity and possible leads to disease progression.

Recently, systemic administration of a human monoclonal antibody directed against cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on circulating T cells in patients with chronic lymphocytic leukaemia (CLL) has been considered. Also, CLL cells have been shown to express CTLA-4, increased levels of which in the leukaemic compartment are a predictor of good clinical outcome. Since both CLL and Treg microenvironment cells can be targeted by the CTLA-4 blocking antibody in this immunotherapy approach, the investigation of the functional effect of CTLA-4 blockade on CLL cells might be of potential clinical relevance. The main aim of this study was to examine the effect of CTLA-4 blockade on proliferation activity and apoptosis of CLL cells in patients with low and high CTLA-4 expression. We found that in the high CTLA-4-expressing CLL group, CTLA-4 blockade on the CLL cell surface resulted in a significant increase in the median percentages of Ki67+ cells and a tendency to decrease in the proportion of apoptotic cells. In contrast, in the low CTLA-4 expressors, CTLA-4 blockade did not affect the proliferation activity or the frequency of apoptosis. This study reports for the first time the different effect of CTLA-4 blockade on CLL cells in CLL patients depending on the levels of CTLA-4 expression. CTLA-4 blockade seems to induce pro-survival signals in leukaemic cells from CLL patients exhibiting high CTLA-4 expression, suggesting that an immunotherapy approach based on the systemic use of monoclonal anti-CTLA-4 antibodies could be an unfavourable strategy for some CLL patients.

Epimutation of the Tumor Suppressor Mechanism in 13q14.3 Involves Monoallelic Expression, Non-Coding RNA Genes and Deregulation of NFkB Signalling

6561 Background: B-cell chronic lymphocytic leukemia (CLL) cells are arrested in G0/G1 phase of the cell cycle and are resistant to programmed cell death, hypothesized to contribute to the resistance of CLL cells to standard chemotherapy with curative intent. Methods: Mec-2 cells and Wac-3 cells are CLL cells that have been shown to be resistant to fludarabine and rituximab. We tested a novel enzyme inhibitor’s ability to render CLL cells sensitive to fludarabine and rituximab. Results: BIBB515, a lanosterol synthase inhibitor, at a concentration of 10μM, was able to reduce the cell viability from 82% in controls to 65% after 72 hours. Fludarabine 10μM alone did not reduce the cell viability, 82 % in controls compared to 80%. BIBB515+ fludarabine treatment for 72 hours, at the dose of 10μM each decreased the cell viability to 37%. Cell proliferation by MTT assay was 0.66±0.010 in control compared to 0.37±0.01 in BIBB515+fludarbine and 0.21±0.01 in BIBB515+ fludarabine+ rituximab. There is a 68% down-regulation of cell proliferation using this treatment. There was a two fold induction of CD 20 with combination treatment, and BIBB515 treatment. The mechanism of cell death in the combination treatment of BIBB515 and fludarabine may be due to the up regulation of cell surface marker CD-20. WAC-3 is another CLL cell line that is sensitive to fludarabine, and resistant to rituximab. BIBB515 sensitizes WAC-3 cells to CD 20 antibody rituximab. There is a 68.7% decrease in cell proliferation with combination treatment of BIBB515 and rituximab. Proliferation of Mec-2 cells were inhibited by 60µM and 30µM terbinafine. Ro-48-8071, showed dose-dependent activity, alone or in combination to fludarabine was seen to induce cell death in Mec-2 cells. Fludarabine alone did not have any effect on these cells. Conclusions: Inhibitors of the mevalonate pathway make resistant CLL cells sensitive to current chemotherapeutic agents. Exploiting this mechanism could alter the current treatment regimens, leading to control of the disease in advanced stages by either inducing the leukemic cells to be static or to regress. This strategy may also limit the toxicities involved with chemotherapy.

Ibrutinib and Venetoclax Target Distinct Subpopulation of CLL Cells: Rationale for Drug Combination and Implication of Minimal Residual Disease Eradication

Introduction. Despite the progress in treatments for chronic lymphocytic leukemia (CLL) in recent years, disease relapse or refractoriness to therapy still occur, thus highlighting the unmet clinical need to find new therapeutic targets for developing alternative drugs. In this context, focal adhesion kinase (FAK) revealed to play a key role in cancer biology, especially in solid tumors. Although knowledge about FAK in hematological malignancies is still limited, some works have begun to show a possible involvement of FAK in the pathogenesis and evolution of CLL. Therefore, carrying on investigation into FAK in CLL may pave the way for the development of new alternative therapies. In this scenario, considering the great prognostic value of TP53 genetic aberrations in CLL and previous evidence of a reciprocal regulation between FAK and p53 in solid tumors, we aimed to determine whether a functional relationship between these two proteins exists also in CLL.Methods. CD19+/CD5+ cells from treatment-naïve CLL patients and normal B cells from healthy donors were purified through density gradient centrifugation. Basal protein expression of FAK and p53 was quantified by western blotting (WB) in 45 CLL patients and 11 controls. Cytoplasm-nucleus protein extraction was performed in 11 CLL patients and then FAK and p53 subcellular distributions were analyzed by WB. This last aspect was also investigated through immunofluorescence (IF) in cells from 2 CLL patients. Eventually, p53 expression in CLL cells after 3 hours of treatment with 5μM of the FAK inhibitor defactinib was assessed by WB in 12 CLL samples.Results. Our WB analysis revealed a significant overexpression of wild type p53 protein (p<0.01) and a higher activation of FAK (in terms of phosphorylation at Y397 residue – pFAK-Y397, p<0.001) in CLL than in normal B cells. Moreover, we found a significant correlation between p53 and FAK protein expression levels in CLL cells (p<0.05, r=0.41). Additionally, by WB and IF, we demonstrated that both FAK and p53 can be present in both cytoplasm and nucleus of CLL cells, with a significant higher presence of p53 (p<0.0001) and pFAK-Y397 (p<0.001) within the nucleus. Accordingly, preliminary results from immunofluorescence revealed that FAK and p53 co-localize in both cytoplasm and nucleus of CLL cells. Adopting a loss-of-function approach, we further demonstrated that FAK inhibition could reduce by 20% the protein expression of p53 in CLL cells (p<0.05).Conclusions. Overall, this study reported some important insights supporting the possible existence of a reciprocal regulation between FAK and p53 also in CLL. At the same time, our results opened the way to consider the role of p53 in CLL biology under a new perspective, suggesting that p53 might have a more complex function in the CLL context than its classical tumor-suppressive activity. In this regard, continuation of this research line could potentially unveil a novel druggable molecular axis in CLL biology.

Clinical Progression of Chronic Lymphocytic Leukemia (CLL) Is Characterized By a Progressive Increase of the Exhausted T-Cell Phenotype and Immunosuppression Induced By Leukemic Cells

Cytokine and T-Cell Phenotypic Changes Upon In Vivo Ibrutinib Therapy For CLL – Targeting Both CLL Cells and The Tumor-Microenvironment

Skin Cancers Among Chronic Lymphocytic Leukemia (CLL) Patients - the Effect of UV Radiation and CLL Clinical Characteristics

On March 20, 2008, bendamustine HCl (Treanda, Cephalon, Inc.) was approved by the U.S. Food and Drug Administration (FDA) for treatment of chronic lymphocytic leukemia (CLL). This approval was based on a randomized, multicenter trial comparing the test drug ( n = 162) to chlorambucil ( n = 157) as a

Introduction: Escape from immunosurveillance is a hallmark of chronic lymphocytic leukemia (CLL) cells. In the protective niche of lymphoid organs, leukemic cells suppress the ability of T lymphocytes to form the immune synapse (IS), thereby hampering T-cell mediated anti-tumoral activities. By binding its cognate receptor PD-1 at the surface of T lymphocytes, the inhibitory ligand PD-L1, which is overexpressed in CLL cells, mediates the T-cell suppressive activities of CLL cells. However, the molecular mechanism underlying PD-L1 overexpression in CLL cells remains unknown. We have previously reported a defective expression of the pro-apoptotic and pro-oxidant adaptor p66Shc in CLL cells, which is causally related to an impairment in intracellular reactive oxygen species (ROS) production and to the activation of the ROS-sensitive transcription factor NF-κB. The fact that PD-L1 expression is regulated by NF-κB suggests a mechanistic relationship between p66Shc deficiency and PD-L1 overexpression in CLL cells. Methods: 62 treatment-naive CLL patients and 43 healthy donors were included in this study. PD-L1 and p66Shc expression was quantified in B cells by flow cytometry and qRT-PCR. IS architecture and local signaling was assessed by flow cytometry and confocal microscopy. CD8+ cell killing activity was assessed by flow cytometry. Results: Here we show that residual p66Shc expression in leukemic cells isolated both from CLL patients and from the CLL mouse model Eμ-TCL1 inversely correlated with PD-L1 expression. We also show that the PD-L1 increase prevented leukemic cells from forming ISs with T lymphocytes. Reconstitution of p66Shc, but not of a ROS-defective mutant, in both CLL cells and the CLL-derived cell line MEC-1, enhanced intracellular ROS and decreased PD-L1 expression. Similar results were obtained following treatment of CLL cells with H2O2 as exogenous source of ROS, that normalized PD-L1 expression and recovered IS formation. Discussion: Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to enhance PD-L1 expression and provides a mechanistic basis for the suppression of T cell-mediated anti-tumoral functions in the immunosuppressive lymphoid niche.

In Vitro and in Vivo Evidence of an Anti-Angiogenic Effect of Lenalidomide in Chronic Lymphocytic Leukemia