PhD candidate BC Cancer Research Centre/ University of British Columbia Vancouver, British Columbia, Canada
Introduction: Previous research on APOBEC3B (A3B) in multiple myeloma (MM) has predominantly focused on APOBEC mutation signatures. However, the role of A3B mRNA expression level and its impact on MM cellular characteristics remains poorly understood. This study aims to investigate the prognostic significance of A3B mRNA expression in MM progression and explore its downstream effects through in vitro experiments.
Methods: The prognostic significance of A3B mRNA expression level in the CoMMpass dataset was investigated using Univariate Cox regression and Kaplan-Meier Curve analysis. Additionally, a pathways analysis (KEGG/REACTOME) was conducted to identify biological functions that were significantly overrepresented in genes correlated with A3B expression. Lentiviral knockdown (KD) and overexpression (OE) of A3B were performed in MM cell lines, followed by RNA-seq and pathway analysis to identify shared pathways through parallel analysis. The obtained results were subsequently tested and validated in vitro.
Results: A3B expression level showed the highest hazard ratio (HROS:2.60; HRPFS:2.32, p< 0.001) among all APOBECs in the MMRF dataset, highlighting the need for functional analysis in MM cells. Genes associated with cell cycle, G1 to S phase transition, and DNA repair response pathways were significantly enriched in the downregulated and upregulated genes in A3B KD (AMO1, L363, LP1, EJM, U266B1) and OE (U266B1, OPM2) MM cell lines, respectively. These findings were validated in the MMRF dataset, showing a significant overrepresentation of these pathways in genes positively correlated with A3B transcript levels and overexpressed in A3B-high (4th quartile) patients versus A3B-low (1st quartile) patients. We showed that A3B KD significantly decreased the proliferation rate and particularly inhibited the G1 to S phase transition in the BrdU/PI assay, resulting in G1 to S arrest. The level of DNA double strand breaks (p-H2AX-S139), single strand DNA breaks (p-S33-RPA32), and deaminase activity (apurinic/apyrimidinic site levels) were quantified in MM cell lines revealing a reduction in A3B KD cells versus cells transduced with empty vector/scrambled. We conducted further investigations to assess the impact of A3B expression levels in patients with Del17p (DNA repair deficiency). Our findings demonstrate that patients carrying del(17p)(CCF>20%) and exhibiting high A3B mRNA expression levels (4th quartile) display a significant (p < 0.05) enrichment of APOBEC-induced genomic mutations, along with a worse prognosis (median PFS: 500 days), in comparison to patients with intermediate (2nd and 3rd quartile) (median PFS: 1000 days) and low (1st quartile) A3B expression levels (median PFS: 1900 days).
Conclusions: These findings highlight the substantial involvement of A3B in cell proliferation and genomic instability in MM. This identifies A3B as a promising candidate for targeted interventions aimed at mitigating DNA damage and genomic instability in MM cells, particularly among patients with del(17p).
