The Role of APOBEC3A and APOBEC3B in the Development of APOBEC Mutational Signatures and Resistance to CDK4/6 Inhibitors in ER-Positive, HER2-Negative Breast Cancer

Introduction - The mutagenic mechanisms driving cancer evolution generate specific mutational signatures. One important signature is attributed to the activity of APOBEC proteins, which act as endogenous sources of mutations and contribute to the development and progression of cancer. APOBEC signatures are associated with poor prognosis and resistance to anti-cancer therapies, but they are also linked to high tumor mutational burden and enhanced immunotherapy response. In breast cancer, they are highly concentrated in the HER2-enriched subtype and have been associated with resistance to endocrine therapy and CDK4/6 inhibitors. Among APOBEC proteins, APOBEC3A and APOBEC3B are primarily implicated in the development of APOBEC signatures in breast cancer. However, the relative contributions of these proteins are not fully understood, and it remains unclear whether the high expression of these proteins is sufficient to induce APOBEC mutagenesis. Objectives - This project aims to investigate whether the overexpression of APOBEC3A or APOBEC3B in breast cancer is associated with the development of APOBEC mutational signatures, resistance to CDK4/6 inhibitors, and whether it is correlated with the HER2-enriched subtype. Methods - A preclinical study was conducted using ER-positive HER2-negative breast cancer cell lines. In the first part of the study, CDK4/6 inhibitor-sensitive and -resistant cell lines were cultivated to assess their APOBEC3A and APOBEC3B expression, PAM50 molecular intrinsic subtype, and APOBEC signatures. In the second part, cell lines overexpressing APOBEC3A and APOBEC3B were generated using a recombinant lentiviral system. These genetically modified cell lines were created to evaluate any change in sensitivity to CDK4/6 inhibitors and PAM50 intrinsic molecular subtype and assess the emergence of APOBEC signatures. Results - CDK4/6 inhibitor-resistant cell lines did not show high expression of APOBEC3A or APOBEC3B, except for the ZR751 cell line, which exhibited elevated APOBEC3B levels and a HER2-enriched subtype. In contrast, cell lines engineered to overexpress APOBEC3A or APOBEC3B did not demonstrate a significant resistance to CDK4/6 inhibitors or a shift to a HER2-enriched profile within the short timeframe studied. Longer-term experiments after extended culturing and the collection of data on APOBEC signatures are needed to assess the full mutagenic potential of APOBEC3A and APOBEC3B proteins and their link to CDK4/6 inhibitors resistance.