Improved Modifications Of Nitric Oxide Donating Aspirin (NO-ASA): Getting a Grip On TP53 Mutated High-Risk Chronic Lymphocytic Leukemia (CLL)

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.

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.

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.

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.

Hallek:Janssen: Research Funding; Gilead: Research Funding; Roche: Research Funding. Kreuzer:Roche: Honoraria; Mundipharma: Honoraria.