Taken together, Trichostatin A appears to act via a dual anti-HDAC/Wnt mechanism with a high selectivity and efficacy in CLL and therefore warrants further investigation.

In actuality, chronic lymphocytic leukaemia (CLL) remains an incurable haematopoietic malignancy of high prevalence amongst elderly populations in the West.

Introduction Chronic lymphocytic leukemia (CLL) shows a conspicuous inhomogeniety in the clinical course among patient subgroups with pronounced genetic aberrations. Some patients have a rapidly fatal disease despite therapy, which is accompanied by the incidence of deletion of chromosome 17p and mutation of TP53. Searching for new therapeutic options we investigate the cytotoxic effects and mechanistically opportunities of NO-ASA on CLL cells. Methods Primary cells were isolated from peripheral blood of CLL patients or healthy individuals. Primary CLL cells with or without TP53 mutation, as well as PBMCs and B-cells of healthy volunteers were tested in a luminometric assay (ATP content) after 24 h NO-ASA incubation. Caspase-dependency was determined in a luminometric assay after 6 h (n=5) as well as in immunoblot-analysis (Caspase 3, PARP; n=3) after 24 h. Influence on Btk- and NF-κB pathways was investigated after 3 h via immunoblot-analysis. Additional, expressions of NF-κB target genes (BCl-2, MCl-1 and CFLAR) were investigated via qRT-PCR after 4 h. Results NO-ASA effectively reduced ATP content in CLL cells from a mixed patient population (LD50 4.34 µM). Primary CLL cells from TP53 mutated patients (n=5) showed a slightly increased LD50 of 25.56 µM, which was still significantly lower than for healthy PBMCs (LD50 63.72 µM, n=5). In addition a concentration-dependent activation of the caspase cascade could be shown compared to untreated control (p<0.001) via luminometric assay as well as a visual apoptosis induction via western blot analysis. Phosphorylation of the NF-κB p65 subunit at Ser 536 was decreased by NO-ASA to ≈58% (n=3), while NO-ASA had no impact on BtK-Phosphorylation status (n=3). The relative expression of BCl-2 and CFLAR is significantly reduced (p<0.05, n=3) by NO-ASA, although MCl-2 was overexpressed (p< 0.001) under treatment’s influence. Conclusion NO-ASA shows potent reduction of ATP content in primary CLL cells. TP53 mutated patients are about three fold more sensitive towards NO-ASA treatment as PBMCs derived from healthy individuals. The cytotoxic effect depends on caspase-activation and might be influenced by inhibition of NFkB-pathway without impact on BtK-pathway. Overexpression of MCl-2 could present an escape-mechanism. Hence, NO-ASA is worth further evaluation as a treatment for poor prognosis patients unresponsive to conventional CLL treatment regimens. Disclosures: Hallek: Janssen: Research Funding; Gilead: Research Funding; Roche: Research Funding. Kreuzer:Roche: Honoraria; Mundipharma: Honoraria.

High progression and resistance to chemoimmunotherapies is the challenge in treatment of TP53 mutated patients. Para-nitric oxide releasing-acetylic salicylic acid (para-NO-ASA) has been shown to possess antineoplastic properties in CLL, but features an insufficient therapeutic index and a low efficacy against TP53 mutated CLL cells. Therefore, the para-NO-ASA was modified and the effect of these newly developed compounds in vitro and in vivo was investigated. Three of the synthesized derivatives effectively induced apoptosis with a high selectivity on CLL cells. This antineoplastic effect was independent of the TP53 mutation status. Derivative B9 demonstrated good tolerability and a strong anti-tumor efficacy in the xenograft mouse with a maximal tumor inhibition rate of 65%. Phosphorylation of the NFκB p65 subunit was significantly reduced by 10 M B9 and B13 by around 75%, while 20 M NO-aspirin could not induce significant reduction. The same applies to the reduction of translocation of NFκB p65 subunit to the nucleus. In addition, expressions of the NF-κB target genes BCL-2, CFLAR and BTK were clearly shut down after incubation with each substance. These results show arresting features of three newly developed derivatives making them promising compounds for high-risk CLL therapy.