Abstract 2384: Investigating mechanisms of tolerance to APOBEC3B upregulation in cancer

The APOBEC proteins are a family of deaminases that convert cytosine to uracil in DNA or RNA. Recent genomic studies designed to uncover mutational processes and drivers of heterogeneity in cancer have revealed that an APOBEC mutational signature is found in over half of all human cancers. As APOBEC3B (A3B) has both preferential deamination activity for cytosines in TCW (W: A/T) motifs in DNA and is upregulated across many cancer types, it is thought to be the predominant source of this mutational signature found in cancer. Previous work has shown that overexpression of A3B in the immortalized HEK293 cell line results in a p53-dependent growth delay, suggesting that increases in A3B may not be tolerated in non-tumorigenic cells. Furthermore, we have shown that this A3B-induced growth delay is dependent on the level of A3B activity and the further processing of the uracil lesions it creates. This suggests that there may be mechanisms, such as p53 loss or DNA damage response (DDR) signaling, that allow the upregulation of A3B expression and activity observed in tumors. We set out to identify DDR processes involved in A3B tolerance in cancer, using non-small cell lung cancer (NSCLC) cell lines, as lung tumors have a strong APOBEC mutational signature. In contrast to what is observed in the HEK293 cells, overexpression of exogenous A3B does not substantially impair the proliferative capacity of NSCLC lines, regardless of TP53 status, suggesting that p53 loss is not the only determinant responsible for A3B tolerance. To investigate additional genes that may be involved in allowing upregulation of A3B, an siRNA library was used to screen for DDR genes that, when lost, restore the growth of HEK293 cell lines overexpressing A3B. The top genes identified as rescuing growth are predominantly involved in double-strand break repair; and these are being further characterized in both HEK293 and NSCLC lines to elucidate potential mechanisms by which they are related to A3B upregulation in tumors. The screen also revealed genes that may be synthetic lethal with elevated levels of A3B. In particular, Checkpoint kinase 1 (CHEK1), which has previously been shown to sensitize cells to high levels of APOBEC3A and A3B, was identified. Synthetic lethal genes are also being explored to establish their interactions with A3B and the potential role they play in the repair of uracil lesions. In conclusion, we have identified DDR genes that, when lost, rescue an A3B-induced growth del