plx-4720 and Leukemia--Lymphocytic--Chronic--B-Cell

Pharmacological inhibition of the potassium channel Kv1.3 has been shown to selectively kill B cells from patients with chronic lymphocytic leukemia (B-CLL). Here we aimed to biophysically characterize and compare Kv1.3 channel activity in B cells isolated either from healthy subjects or patients and investigated the mechanism accounting for the increased protein expression in B-CLL cells.. Kv1.3 activity was measured by patch clamp, while expression of the channel protein was assessed by Western blot and FACS analysis. B-CLL cells were co-cultured with mesenchymal stromal cells (MSC) and Kv1.3 inhibitor-induced apoptosis was assessed.. We demonstrate that Kv1.3 is highly expressed and is more active at resting membrane potential in human B-CLL cells than in healthy cells. Channel expression in pathologic cells decreased by the B-RAF kinase inhibitor PLX-4720, while it increased with Doxazosin, an α1-adrenoceptor antagonist. Kv1.3 inhibitors induced death in B-CLL cells also when co-cultured with MSC.. Our results contribute to the characterization of B-CLL cells, as it shows that upregulation of Kv1.3 in pathologic B lymphocytes is linked to the oncogenic B-RAF signalling. We also conclude that Kv1.3 inhibitors represent a valuable tool to induce apoptosis of B-CLL cells even in the presence of MSC.

Topics: Apoptosis; B-Lymphocytes; Cell Proliferation; Cells, Cultured; Cinnamates; Coculture Techniques; Cyclopropanes; Doxazosin; Humans; Indoles; Jurkat Cells; Kv1.3 Potassium Channel; Leukemia, Lymphocytic, Chronic, B-Cell; Membrane Potentials; Mesenchymal Stem Cells; Proto-Oncogene Proteins B-raf; Signal Transduction; Sulfonamides; Up-Regulation

BRAF mutations have been shown to occur at a high frequency in melanoma and thyroid cancer, but also at lower frequencies in hematological malignancies. To assess the potential role of BRAF, we have sequenced exons 11 and 15 of BRAF in 138 cases with chronic lymphocytic leukemia (CLL) and 32 cases of B-cell prolymphocytic leukemia (B-PLL). We found an incidence of BRAF mutations of 2.8% in CLL (4/138), while no cases with B-PLL showed BRAF mutations. The analysis of a cohort of patients with fludarabine-refractory disease (n = 87) showed no increase in the mutation incidence, suggesting that this mutation is not selected for during the disease progression. A limited analysis of the effect of BRAF inhibition in primary CLL cells showed no cell death induction in CLL samples with and without BRAF mutations. Our analysis suggests that BRAF mutations occur at a low frequency in CLL. The pharmacological inhibition of MEK/ERK signaling using the mutant BRAF inhibitor PLX4720 showed no effect on viability in vitro in CLL cases.

Topics: Antineoplastic Agents; Apoptosis; Exons; Extracellular Signal-Regulated MAP Kinases; Humans; Indoles; Leukemia, Lymphocytic, Chronic, B-Cell; Mitogen-Activated Protein Kinases; Mutation; Niacinamide; Phenylurea Compounds; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Signal Transduction; Sorafenib; Sulfonamides; Treatment Outcome