Investigating acute cellular responses to elevated APOBEC3B expression

Abstract

The cytosine deaminase APOBEC3B (A3B) functions as part of innate immunity by mutating viral DNA and preventing replication. Large-scale genome sequencing studies and examination of expression levels have identified mutations associated with elevated A3B activity in a number of human cancers. However, the mechanisms that allow cancer cells to cope with elevated A3B expression and activity remain unclear. To identify these mechanisms, the effects of A3B overexpression were characterised in normal and cancer cell line models. In several non-small cell carcinoma (NSCLC) cell lines, A3B overexpression is largely tolerated, consistent with high levels of A3B found in lung cancer patient tumours. In contrast, the non-transformed HEK293 cell line undergoes growth inhibition in response to A3B overexpression, dependent on both the deamination activity of the enzyme and the subsequent activity of uracil DNA glycosylase on the aberrant uracil. These findings suggest that defects in repair pathways downstream of uracil excision could be important for cells to tolerate A3B-induced DNA damage. A DNA damage and repair RNAi library was screened to identify genes that, when lost, would either rescue or enhance growth inhibition in HEK293 cells overexpressing A3B. The DNA helicase RECQL and the DNA damage sensing proteins PARP1 and PARP2 were identified as potential hits that rescued the growth defect, but failed to validate in follow-up experiments. By contrast, inhibition of checkpoint kinase CHK1 function, either by RNAi targeting CHEK1 or pharmacological inhibition of ATR or CHK1, sensitises HEK293 cells to A3B overexpression. Interestingly, this combination has minimal effects in several NSCLC lines. CHK1 phosphorylation, an indicator of kinase activation, is increased with A3B overexpression in the HEK293, suggesting A3B expression enhances replication stress. Additionally, ATR and CHK1 inhibitors decrease A3B activity, but not expression, in cells suggesting checkpoint activation regulates A3B activity. These findings have highlighted potential roles of DNA damage response and CHK1 signalling in protecting cells from aberrant A3B activity and how deregulation of these may contribute to elevated A3B expression in cancer.