Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells.

Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD(+)-independent HDACs are an established therapeutic target, the relevance of NAD(+)-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+)-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+) levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD(+)-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.

Apoptosis of B-cell chronic lymphocytic leukemia cells induced by a novel BH3 peptidomimetic.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in human adults of the Western world and no definitive cure is yet available. The disease is characterized by accumulation of clonal malignant B lymphocytes resistant to apoptosis. Strategies to hit the anti-apoptotic drift of the Bcl-2 family in B-CLL cells are being explored. A novel peptidomimetic based on the BH3 domain of the pro-apoptotic protein Bim and recently shown to exert significant apoptotic activity on acute myeloid leukemia cells, both in vitro and in vivo, was assayed on ex-vivo derived leukemic cells from untreated B-CLL patients (n = 7). We found that this peptide, named 072RB, induced apoptosis of B-CLL samples at a concentration that does not affect viability of peripheral and bone marrow derived lymphocytes from healthy donors. Apoptosis was demonstrated by activation of Bak and Bax, externalization of plasma membranes phosphadydilserines, appearance of hypodiploid events in DNA flow cytometry histograms and was accompanied by dissipation of the mitochondrial transmembrane potential. Before the onset of marked apoptotic signs a progressive decline of the relevant anti-apoptotic proteins Bcl-X(L) and Mcl-1 could be observed. The negative control peptide 072RBL94A was ineffective for B-CLL cells, supporting the sequence specificity of 072RB activity. No relationship was found between responsiveness to 072RB and Mcl-1/Bcl-X(L) basal levels or decrease magnitude, possibly because of the limited sample size of the study. Altogether, we demonstrate that 072RB induces significant apoptosis of B-CLL cells subsequent to Bcl-X(L) and Mcl-1 downregulation.

Clonal granulocytes in polycythaemia vera and essential thrombocythaemia have shortened telomeres.

Summary The purpose of this study was to evaluate telomere length in peripheral blood granulocytes and mononuclear cells collected from 22 women with polycythaemia vera (PV) and essential thrombocythaemia (ET). PV and ET are chronic myeloproliferative diseases whose heterogeneity of stem cell origin and clonal development has been established through analysis of X-chromosome inactivation patterns. The results from clonality assay and determination of telomere length show that only clonal granulocytes have shortened telomeres.

High telomerase activity in granulocytes from clonal polycythemia vera and essential thrombocythemia.

Essential thrombocythemia (ET) and polycythemia vera (PV) are chronic myeloproliferative disorders that share the involvement of a multipotent progenitor cell and dominance of the transformed clone over normal hematopoiesis. On the other hand, the heterogeneity of these diseases with respect to clonal development from a common progenitor has been well established. To identify useful prognostic indicators, we analyzed telomerase activity (TA), a known marker of neoplastic proliferation, in granulocytes (PMNs) and mononuclear cells (MNCs) from 22 female patients with ET and PV. Clonality status was determined by investigation of X chromosome inactivation patterns (XCIPs). We found a statistically significant positive correlation between high TA and monoclonal pattern of XCIP. Therefore, our data suggest that the use of multiple tumor markers may contribute to a better understanding of the deregulated physiology of these disorders and provide useful prognostic factors.