Abstract
Although there have been dramatic improvements in the treatment of paediatric ALL in recent years, a significant proportion of these patients still experience relapse. Early treatment responses, indicated by the rate and degree of blast clearance, are good markers for the likelihood of ALL relapse. However, despite their clinical predictive value, the molecular basis underlying these observations remains unknown.
We aimed to elucidate the molecular mechanisms behind treatment resistance in paediatric ALL by analysing cellular responses to IR-induced damage. Using western blotting, we classified 82 paediatric diagnostic ALL samples into two categories on the basis of their activation of apoptosis following DNA damage. We found that 31 ALL tumours had an apoptotic resistant phenotype, failing to cleave PARP1 and caspases 3, 7 and 9 by 24h after ionising radiation (IR), in contrast to 51 tumours classified as apoptotic responsive. Strikingly, we observed that our DNA damage apoptotic response stratification of ALL tumours closely correlated with blast clearance following treatment in ALL patients. Among apoptotic resistant patients, 69% still had more than 25% blasts by morphological assessment on day 8 or 15, and 64% had a blast population that could be detected by molecular minimal residual disease (MRD) at day 28, indicating high risk of relapse. In contrast, in the apoptotic responsive group only 8% had more than 25% blasts at day 8 or 15, and just 6% were positive by MRD at day 28.
Differential baseline expression of specific genes has been previously linked with drug resistance in paediatric ALL. To provide further insight into the biology of resistance to DNA damaging agents we analysed changes in transcriptional profiles following exposure of blast cells to IR in 22 representative tumours, 11 apoptotic responsive and 11 apoptotic resistant. We identified over 300 IR-induced genes that could discriminate between the two categories, including genes involved in cellular proliferation and the regulation of apoptosis. When we considered only the subset with TEL/AML1 fusion, we found that the expression of phospholipase C epsilon (PLCe), which is implicated in the Ras cellular survival pathway, provided powerful discrimination between the apoptotic resistant and responsive ALL tumours. We have subsequently confirmed that PLCe is also differentially expressed at the protein level at 24h after IR between apoptotic resistant and responsive ALLs, regardless of TEL/AML1 status. Finally, we have identified model pre-B ALL cell lines for each of the two response categories, and in these we have shown that Ras is activated in response to DNA damage and associates with PLCe only in the apoptotic resistant cells.
In conclusion, we have shown that differences in cellular apoptotic response to DNA damage closely correlate with the rate of blast clearance in paediatric ALL. Furthermore, we believe that alteration in the Ras survival pathway is one mechanism to account for differential apoptotic responses to DNA damage in both primary ALL tumour cells and ALL cell lines. These findings provide new insight into the factors influencing clinical outcome in ALL patients and are likely to have important implications for the development of future targeted treatments in paediatric ALL.
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