sq-23377 and Leukemia--Lymphocytic--Chronic--B-Cell

Chronic lymphocytic leukemia (CLL) is a hematological neoplasm of CD19-positive mature-appearing B lymphocytes. Despite the clinical success of targeted therapies in CLL, the development of resistance diminishes their therapeutic activity. This is also true for the Bcl-2 antagonist venetoclax. We investigated the molecular mechanisms that drive venetoclax resistance in CLL, with a clear focus to provide new strategies to successfully combat it. Activation of CLL cells with IFNγ, PMA/ionomycin, and sCD40L diminished the cytotoxicity of venetoclax. We demonstrated that the metabolic activity of cells treated with 1 nM venetoclax alone was 48% of untreated cells, and was higher for cells co-treated with IFNγ (110%), PMA/ionomycin (78%), and sCD40L (62%). As of molecular mechanism, we showed that PMA/ionomycin and sCD40L triggered translocation of NFκB in primary CLL cells, while IFNγ activated p38 MAPK, suppressed spontaneous and venetoclax-induced apoptosis and induced formation of the immunoproteasome. Inhibition of immunoproteasome with ONX-0914 suppressed activity of immunoproteasome and synergized with venetoclax against primary CLL cells. On the other hand, inhibition of p38 MAPK abolished cytoprotective effects of IFNγ. We demonstrated that venetoclax-resistant (MEC-1 VER) cells overexpressed p38 MAPK and p-Bcl-2 (Ser70), and underexpressed Mcl-1, Bax, and Bak. Inhibition of p38 MAPK or immunoproteasome triggered apoptosis in CLL cells and overcame the resistance to venetoclax of MEC-1 VER cells and venetoclax-insensitive primary CLL cells. In conclusion, the p38 MAPK pathway and immunoproteasome represent novel targets to combat venetoclax resistance in CLL.

Topics: Antineoplastic Agents; bcl-2-Associated X Protein; Bridged Bicyclo Compounds, Heterocyclic; Drug Resistance, Neoplasm; Humans; Ionomycin; Leukemia, Lymphocytic, Chronic, B-Cell; Myeloid Cell Leukemia Sequence 1 Protein; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Topics: Animals; Antigen-Presenting Cells; Antigens, CD; Cancer Vaccines; CD8-Positive T-Lymphocytes; Disease Models, Animal; Humans; Ionomycin; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Depletion; Mice, Inbred NOD; Mice, SCID

Nucleotides (NTPs and dNTPs) have been measured in patient-derived chronic lymphocytic leukaemia (CLL) cells stimulated with TPA plus ionomycin and then exposed to cladribine, fludarabine or deoxycoformycin (1 microM, 16 h). dNTPs were not measurable in 10(8) unstimulated CLL cells which are quiescent. Time-dependent changes in nucleotides in CLL cells showed that the mechanism of action changed as the drug triphosphate accumulated. dCf induced substantial accumulation of dATP in CLL cells in culture, and similar levels of dATP in the same patient during subsequent treatment with dCf. Determination of "metabolic response patterns" of nucleotides in CLL cells treated with drugs might distinguish patients with susceptible and refractory CLL prior to chemotherapy.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cladribine; Humans; Ionomycin; Kinetics; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Nucleotides; Pentostatin; Prognosis; Tumor Cells, Cultured; Vidarabine

Profound immune dysfunction is a constant feature in B-cell chronic lymphocytic leukemia (B-CLL) patients. Immunological abnormalities include hypogammaglobulinemia, impaired immunoglobulin class switching and diminished germinal center formation. This state of immune suppression renders B-CLL patients highly susceptible to infections, which contribute greatly to morbidity and mortality in this disease. Impaired T cell function in B-CLL is well-documented and has been suggested to result from inhibitory effects exerted by malignant B lymphocytes. Because the presence of leukemic cells may represent a major obstacle to efficient T cell activation, T lymphocytes were separated from CLL B cells, stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4h, and then cocultured with autologous leukemic B cells both at a 1:1 ratio or at the same ratio as in vivo for 24-40 h. CLL B cell expression of CD86 and CD95 was markedly upregulated using this approach, whereas CD80 expression was augmented only in a minority of patients; these effects were partially preserved even when preactivated T cells were rechallenged with CLL B cells at the same low T/B cell ratio as that observed in vivo. Finally, CD80 upregulation on CLL B cells appeared to be mainly dependent on CD40L-mediated stimulation, whereas CD86 and CD95 expression was efficiently augmented by soluble factors released by preactivated T lymphocytes. In conclusion, efficient activation of T lymphocytes in B-CLL may be achieved which, in turn, may result in enhanced antigen-presenting capacity and susceptibility to apoptosis of leukemic cells via CD86 and CD95 upregulation, respectively.

Topics: Aged; Aged, 80 and over; Antigens, CD; Apoptosis; B-Lymphocytes; B7-1 Antigen; B7-2 Antigen; CD40 Ligand; Coculture Techniques; fas Receptor; Female; Humans; Immunophenotyping; Ionomycin; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Activation; Male; Membrane Glycoproteins; Middle Aged; T-Lymphocytes; Tetradecanoylphorbol Acetate; Up-Regulation

It has been shown previously that chronic lymphocytic leukemia (CLL) B cells are frozen at different stages of activation with unique requirements for proliferation. Although most B CLL cells express surface IgM, anti-mu antibodies are able to trigger only some of them to proliferate and/or respond to B cell growth factor (BCGF) or interleukin 2 (IL2), as normal B cells. In this report we extend these observations using three different monoclonal antibodies (mAb) to human mu chain (one mitogenic in soluble form for normal B cells, the two others mitogenic only when coupled on Sepharose 4B beads). Cells from only 3 out of 11 B CLL patients proliferated in the presence of either mitogenic anti-mu. When the early events following surface Ig cross-linking, such as calcium mobilization (by flow cytometry on indo-1-labeled cells), were studied all three mAb in soluble form were able to induce a similar increase in the intracellular free calcium concentration ([Ca2+]i); analogous to [Ca2+]i rise after the mitogenic F(ab')2 anti-mu stimulation. This response was seen only in 8 out of the 12 CLL B cells studied. All B CLL cells, however, proliferate in response to a combination of phorbol ester 12,13-dibutyrate (PBt2) and ionomycin. Therefore, three patterns of response to sIg cross-linking by anti-mu could be distinguished: cells from 4 out of 12 cases proliferate and mobilize Ca2+ upon anti-mu triggering (behaving like resting B lymphocytes); in 4 other cases anti-mu lead to Ca2+ mobilization without cell proliferation; in the last 4 cases neither Ca2+ mobilization, IP3 generation (in the one case studied) nor cell proliferation are observed although these cells do proliferate directly in response to growth factors. Moreover, anti-mu stimulation in this group leads to increased proliferation in response to BCGF and IL2 suggesting an anti-Ig signaling independent of inositol phosphate metabolism. These results are interpreted in terms of differential anti-mu signaling on B cells at different stages of activation.

Topics: Antibodies, Anti-Idiotypic; B-Lymphocytes; Calcium; Cell Division; Cross-Linking Reagents; Ethers; Humans; Immunoglobulin mu-Chains; Inositol Phosphates; Interleukin-2; Interleukin-4; Interleukins; Ionomycin; Leukemia, Lymphocytic, Chronic, B-Cell; Phorbol Esters; Receptors, Antigen, B-Cell; Tumor Cells, Cultured