Salvatore Casciaro

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.

APO866 activity in hematologic malignancies: a preclinical in vitro study

To the editor: Nahimana and coworkers have recently reported that the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor APO866 elicited massive cell death in primary leukemia cells and in numerous leukemia/lymphoma cell lines.[1][1] In particular, in 32 primary leukemias (including 12 B-cell

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-XL 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-XL 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-XL and Mcl-1 down-regulation.

Heterogeneity of clonal development in chronic myeloproliferative disorders

Recent reports have suggested a previously unexpected variability in the expression of the dominant neoplastic clone in myeloproliferative disorders (MPD). We evaluated 49 female patients with MPD and informative at the X‐linked androgen receptor (AR) locus to establish the X chromosome inactivation pattern of hemopoietic cells. Whereas in chronic myelogenous leukemia (CML) the granulocytes (PMN) were uniformly of monoclonal origin, a striking heterogeneity of clonal development was found in PMN from patients with other MPD, with up to 50% of them expressing a polyclonal pattern of X inactivation. Am. J. Hematol. 60:158–160, 1999.

N -(4-hydroxyphenyl)retinamide promotes apoptosis of resting and proliferating B-cell chronic lymphocytic leukemia cells and potentiates fludarabine and ABT-737 cytotoxicity

The in vitro effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR, fenretinide) on primary B-cell chronic lymphocytic leukemia (CLL) cells from previously untreated CLL patients were investigated. 4HPR promoted the intrinsic apoptotic pathway by reactive oxygen species (ROS) generation and was accompanied by drop of Mcl-1 protein expression. The latter was not attributable to transcriptional downregulation but to protein degradation mediated by jun N-terminal kinase activation, and likely by NF-kB downregulation and Noxa upregulation. CLL cells stimulated in vitro with CD40L did not increase 4HPR chemoresistance if activation was accompanied by proliferation. Intra-patient analysis confirmed that the proliferating pool of CLL cells was more sensitive to the cytotoxic action of 4HPR than the activated but resting CLL subpopulation. The different 4HPR susceptibility of the two subpopulations was associated with higher Noxa expression in proliferating CLLs. Combination experiments revealed that 4HPR strongly potentiated ABT-737 cytotoxicity, especially in proliferating CLL cells that displayed amplified chemoresistance to ABT-737 alone. Synergic cytotoxicity was also demonstrated in combination with fludarabine, in both resting and stimulated CLL samples. This study entitles 4HPR to be assayed as a chemotherapeutic adjuvant for the treatment of CLL.

Annular pustular psoriasis and systemic lupus erythematosus

A 30-year-olcl woman presented in 1970 with a history of fever, arthralgia, Raynauds phenomenon, and dyspnea. On examination, she disclosed a butterfly rash, lymphadenopathy, and peritoneal and bilateral pleural effusion. There were hemolytic anemia, thrombocytopenia, and a positive lupus erythematosus (LE) cells test. Systemic LE (SLE) was diagnosed and 30 mg/day prednisone and 150 mg/day azathioprine prescribed. Eventually, she developed nephropathy, which was treated with steroid pulses. In 1985 a palmo-plantar eruption was diagnosed as pustolosis, palmaris, and plantaris (PPP) and, though partially cured with topical steroids, relapsed 4-5 days after every steroid pulse. Eventually, a 2 g/day proteinuria and a diffuse pustular eruption developed. Examination revealed annular, erythematous areas with numerous pustules at their edges and erosions and crusts involving the trunk and lower (Fig. 1) and upper limbs. Head and mucous membranes were spared. Nikolskys sign was negative. Erythrocyte sedimentation rate ranged from 65 to 109 mm/hour and proteinuria from 4 to 12 g/dl. C3 was 70 mg/dl (normal values, 88-177) and C4 was 13 mg/dl (normal, 15-45). Antinuclear antibodies were >1:320 with a homogeneous pattern. Absent were anti-dsDNA, anti-ENA, and anti-SSA antibodies. HLA A3 11 B35 CW 4 and DR 4 W53 DQ W1 were found at typing. Histopathology of two annular lesions showed a subcorneal accumulation of neutrophils and cellular debris (Fig. 2) with a typical Kogos spongiform pustule in the underlying acanthotic epidermis (Fig. 3). No acantholytic cells were seen. The upper dermal vessels showed endothelial swelling and luminal obliteration with a polymorphous infiltrate and signs of leukocytoclasia (Fig. 4). Direct immunofluorescence of both lesions showed granular deposits of C3 at the dermoepidermal junction, IgM fluorescent bodies in the

Clonal granulocytes in polycythaemia vera and essential thrombocythaemia have shortened telomeres

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.