Abstract

Defects in cell death signalling might be responsible for treatment failure and relapse in acute leukaemia. In a study on paediatric acute lymphoblastic leukaemia (ALL) we recently reported the importance of intact apoptosis signalling for favourable outcome. Here we addressed this issue in 45 paediatric acute myeloid leukaemia (AML) patient samples. Two key apoptogenic events: cytochrome c release and caspase-3 activation were analysed by flowcytometry in the leukaemia cells after induction of apoptosis by factor deprivation. Release of cytochrome c and consecutive apoptosome formation leads to activation of downstream effector caspases such as caspase-3. Both events were closely correlated to each other in good responding patients (less than 5% blasts in bone marrow or minimal residual disease negativity) and in patients achieving remission. No correlation was found in patients with poor treatment response or patients not reaching remission. Caspases other than caspase-3 acting upstream of mitochondria (e.g. caspase-8) are also involved in initiating or amplifying this complex. By incubation in presence or absence of the pan-caspase inhibitor zVADfmk cytochrome c release was observed to be dependent or independent on upstream caspases. Of note, only cytochrome c released in dependence on amplifying caspases was observed to correlate with activated caspase-3 in patients with good response to treatment or patients without relapse. This indicates that an intact apoptosome function and active amplifying caspases are elementary for favourable outcome in childhood AML. The functional integrity of this important apoptogenic checkpoint is subsumed by the parameter caspase dependent cytochrome c-related activation of caspase-3 (CRACdep). Division of our cohort according to this new paramenter resulted in two groups of 38 CRACdep -positive and 7 -negative patients, the distribution of the values being unrelated to features such as FAB subtype or risk groups. 30 of 32 patients sustaining complete remission for more than one year were CRACdep -positive (Fisher exact test, P= .015). Analysis of survival (N=45, log rank P= .014) and remission duration (N=40, log rank P= .001) revealed a superior outcome for CRACdep -positive patients. This was also observed when considering the high risk patients only. Notably, standard risk stratified patients merely displayed CRACdep -positivity. Thus, the propensity to undergo apoptosis as reflected by intact signalling in leukaemia cells is an important feature for favourable treatment outcome and may serve as additional stratification tool for paediatric AML patients.

Author notes

Disclosure:Research Funding: Research fellowship grant of EHA (Europaen Hematology Association) to LHM.