Upregulation of FLIPs upon CD40 Stimulation - A Novel Inhibititory Mechanism of CD95-Induced Apoptosis in Precursor B-ALL Blasts in Children.

The influence of the microenvironment on the activation status and behaviour of ALL blasts is critical for interactions with the immune system in vivo . The capacity of B cells to respond to CD40-ligand (CD40L) stimulation is critical for their sensitisation to immunological control mechanisms and susceptibility to apoptotic signals. Primary precursor B-ALL blasts (BCP-ALL; n=32) lack CD95-expression (mean±SE; 4.2±0.6% positive cells) and are resistant to apoptosis while significant up-regulation of CD95 is apparent upon CD40-stimulation in BCP-ALL blasts that reaches a plateau after 72 h. Yet, in spite of equivalent CD95-upregulation in ALL blasts (58.3±6.5%; n=17) and normal B cells (59.3±13.1%) specific apoptosis is markedly lower in ALL compared to mature B cells (19.1±3% vs 36.7±5.5%). Resistance to apoptosis in ALL blasts and its reversibility after cycloheximid treatment suggest that anti-apoptotic mechanisms prevent induction of cell death via CD95 ligation in CD40 activated blasts. In accordance, in CD40-activated ALL blasts caspase 8 and 3 activity is not enhanced upon CD95 ligation in contrast to an 1.8±0.3 and 1.7±0.3 fold increase in caspase activity in stimulated normal B cells (n=7), suggesting a block of the apoptotic cascade in BCP-ALLrelatively close to the receptor level. CD40L-activated ALL blasts and normal B cells were submitted to western blot analysis with respect to the molecules associated to the death-inducing signalling complex (DISC). FADD and the zymogen form of caspase-8 are constitutively expressed in both malignant and non malignant B cells with no modulation following CD40 ligation. In contrast, the anti-apoptotic short isoform of the c-FLICE inhibitory protein FLIP S is weakly expressed in naive blasts and B cells, but strongly up-regulated upon 72h CD40-ligation in ALL with only barely detectable levels in CD40-activated normal B cells. We therefore propose, that prolonged induction of the FLIP S expression inhibits the onset of apoptosis despite high CD95 surface expression levels in BCP-ALL blasts. As an additional anti-apoptotic mechanism inhibiting the downstream effector caspases we demonstrated significant upregulation of the inhibitor of apoptosis protein (IAP) survivin in CD40-activated BCP-ALL (n=6) compared to the unstimulated control (632pg/ml±200pg/ml vs 180pg/ml±52pg/ml). Thus, we identified FLIP S as a CD40-regulated upstream anti-apoptotic element and concomitant downstream upregulation of survivin protein expression as critical mechanisms contributing to blast cell resistance to apoptosis.