Federico Chiurazzi

Selective inhibition of PED protein expression sensitizes B-cell chronic lymphocytic leukaemia cells to TRAIL-induced apoptosis

B‐cell chronic lymphocytic leukaemia (B‐CLL) cells fail to undergo apoptosis. The mechanism underlying this resistance to cell death is still largely unknown. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) effectively kills tumour cells but not normal cells, and thus represents an attractive tool for the treatment of cancer. Unfortunately, lymphocytes from B‐CLL patients are resistant to TRAIL‐mediated apoptosis. Thus, we aimed to study the involvement of PED, a DED‐family member with a broad antiapoptotic action, in this resistance. We demonstrate that B lymphocytes obtained from patients with B‐CLL express high levels of PED. Treatment of B‐CLL cells with specific PED antisense oligonucleotides, a protein synthesis inhibitor or HDAC inhibitors, induced a significant downregulation of PED and sensitized these cells to TRAIL‐induced cell death. These findings suggest a direct involvement of PED in resistance to TRAIL‐induced apoptosis in B‐CLL. It also identifies this DED‐family member as a potential therapeutic target for this form of leukaemia.

Effects of preactivated autologous T lymphocytes on CD80, CD86 and CD95 expression by chronic lymphocytic leukemia B cells.

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 4 h, 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.

Combined Factor V and Factor VII Deficiency

A patient suffering from cardiochalasia was found to be partially deficient in both coagulation factors V and VII. No bleeding tendency had been noticed. A family study showed that the father had fact

The effect of FK506 on transforming growth factor β signaling and apoptosis in chronic lymphocytic leukemia B cells

Loss of response to transforming growth factor-β (TGF-β) is thought to contribute to the progression of chronic lymphocytic leukemia. This study shows that chronic lymphocytic leukemia cells do indeed escape the homeostatic control of TGF-β. Background Loss of response to transforming growth factor-beta (TGF-β ) is thought to contribute to the progression of chronic lymphocytic leukemia. Recent findings of over-activation of the TGF-β signal in FKBP12-knockout mouse prompted us to investigate whether FK506, the canonical ligand of FKBP, can activate the TGF-β signal in chronic lymphocytic leukemia. Design and Methods We studied 62 chronic lymphocytic leukemia samples from patients with Rai/Binet stage 0 to 4 disease. The TGF-β signal was investigated by western blotting and flow cytometry. The levels of Bcl2-family members and death-associated-protein kinase were also investigated by western blotting, whereas apoptosis was studied in flow cytometry. Down-modulation of FKBP12 was obtained by gene silencing with short interfering RNA. Results Twenty-two out of 62 chronic lymphocytic leukemia samples were sensitive to TGF-β-induced apoptosis. All but two of the responsive samples underwent apoptosis also when cultured with FK506, but not with cyclosporine. Thirteen samples that were not sensitive to TGF-β were sensitive to FK506. Overall, response to FK506 occurred in 33 samples. FK506 induced Smad2 phosphorylation and nuclear translocation. Accordingly, death-associated-protein kinase, a transcriptional target of Smad, was induced. At the same time, Bcl-2 and Bcl-xL levels decreased whereas the levels of Bim and Bmf increased. A loss of mitochondrial membrane potential preceded caspase activation and cell death. FK506 removed FKBP12 from its binding to the TGF-β-receptor. FKBP12 release activated the receptor-kinase activity as suggested by the enhanced levels of phospho-Smad found in cells depleted of FKBP12. Conclusions Our study shows that most chronic lymphocytic leukemia cells escape the homeostatic control of TGF-β and that FK506 restores the TGF-β signal in a proportion of non-responsive samples. We demonstrated that FK506 activates TGF-β receptor I kinase activity in chronic lymphocytic leukemia, which transduces apoptosis by a mitochondrial-dependent pathway.

Clinical and phenotypic features of CD5-negative B cell chronic lymphoproliferative disease resembling chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) B cells are phenotypically identified by surface expression of CD5 and CD23 antigens. Infrequently, patients with a monoclonal B cell lymphocytosis clinically resembling classic B-CLL have been found to harbor leukemic B cells lacking expression of the CD5 antigen. Little information is available concerning such CLL-like lymphoproliferative syndromes. Here, we provide phenotypic and clinical characteristics of 13 patients with CD5-negative chronic lymphoproliferative disorders selected from among 400 B-CLL patients followed up at a single academic center. Phenotypic analysis was carried out by flow cytometry using a broad panel of monoclonal antibodies including activation, costimulatory, adhesion, and growth factor receptor molecules. Moreover, intracellular staining and stimulation experiments were performed to investigate whether CD5 antigen was either retained in the cytoplasm of clonal B cells or not expressed due to defective cellular activation, respectively. Overall, CD5-negative leukemic cells were found to express significantly different levels of several membrane molecules, including CD95, CD69, CD23, CD25, CD80, and CD20, compared to “classic” CLL B cells. CD5 antigen was not detected in the cytoplasm of CD5-negative clonal B cells, nor could it be induced following in vitro activation. CD3+ T cell proportions were found to be less affected in CD5-negative patients than in classic B-CLL. Although these data suggest that CD5-negative clonal B cells are phenotypically different from classic B-CLL, clinical outcomes were similar to those shown by B-CLL patients, with most of the patients experiencing a long-lasting disease requiring chemotherapeutic intervention at some time during the disease course.

Predictors of refractoriness to therapy and healthcare resource utilization in 378 patients with primary autoimmune hemolytic anemia from eight Italian reference centers

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Role of ZNF224 in cell growth and chemoresistance of chronic lymphocitic leukemia

Chronic lymphocytic leukaemia (CLL) is associated with apoptosis resistance and defective control of cell growth. Our study describes for the first time a critical role in CLL for the KRAB-zinc finger protein ZNF224. High ZNF224 transcript levels were detected in CLL patients with respect to control cells. Moreover, ZNF224 expression was significantly lowered after conventional chemotherapy treatment in a subset of CLL patients. By in vitro experiments we confirmed that ZNF224 expression is suppressed by fludarabine and demonstrated that ZNF224 is involved in apoptosis resistance in CLL cells. Moreover, we showed that ZNF224 positively modulates cyclin D3 gene expression. Consistently, we observed that alteration of ZNF224 expression leads to defects in cell cycle control. All together, our results strongly suggest that in CLL cells high expression level of ZNF224 can lead to inappropriate cell growth and apoptosis resistance, thus contributing to CLL progression. Targeting ZNF224 could thus improve CLL response to therapy.

Ultrasonography-driven combination antibiotic therapy with tigecycline significantly increases survival among patients with neutropenic enterocolitis following cytarabine-containing chemotherapy for the remission induction of acute myeloid leukemia

Neutropenic enterocolitis (NEC) is an abdominal infection reported primarily in patients with acute myeloid leukemia (AML) following chemotherapy, especially cytarabine, a notable efficacious cytotoxic agent for AML remission. Specific data regarding the impact of different cytarabine schedules and/or antibacterial regimens for NEC are sparse. The aim of the study was to identify the predictors of outcome within 30 days of NEC onset. NEC episodes were retrospectively pinpointed among 440 patients with newly diagnosed AML hospitalized in our Institution, over a 10‐year period, for receiving chemotherapy protocols with 100–6000 mg/m2 daily of cytarabine. Two subgroups, survivors versus nonsurvivors, were compared by using logistic regression analysis. NEC was documented in 100 of 420 (23.8%) analyzed patients: 42.5% had received high‐dose cytarabine, whereas 19% and 15% intermediate‐dose and standard‐dose cytarabine, respectively (P < 0.001). The 30‐day NEC attributable mortality rate was 23%. In univariate analysis, antileukemic protocols containing robust dosages of cytarabine were significantly associated with high mortality (P < 0.001); whereas, standard‐dose cytarabine and prompt initiation (at the ultrasonographic appearance of intestinal mural thickening) of NEC therapy with antibiotic combinations including tigecycline were significantly associated with low mortality. In multivariate analysis, high‐dose cytarabine‐containing chemotherapy was the independent predictor of poor outcome (odds ratio [OR]: 0.109; 95% confidence interval [CI]: 0.032–0.364; P < 0.001), whereas ultrasonography‐driven NEC therapy with antibiotic regimens including tigecycline was associated with a favorable outcome (OR: 13.161; 95% CI: 1.587–109.17; P = 0.017). Chemotherapy schedules with robust dosages of cytarabine for AML remission are associated with a high rate of NEC incidence and attributable. Vigorous antibacterial therapy, triggered off pathologic ultrasonographic findings, with drug combinations which have broad antimicrobial coverage and good gut penetration, specifically those also including tigecycline, may be effective in improving 30‐day survival rate after NEC onset.