APOBEC3B (A3B) is a DNA cytosine deaminase and catalyzes cytosine deamination, resulting in mutations in genomic DNA. A3B is aberrantly expressed in a variety of cancers and considered to be a source of genomic mutations that contribute to cancer progression and metastasis. However, the mechanisms through which A3B expression is dysregulated in cancer cells are not fully elucidated. Here we report that the classical NF-κB pathway plays a crucial role in the transcriptional regulation of A3B in various cancer cells, including lymphoid malignancies. PMA, a strong activator of PKC, induces A3B at both mRNA and protein levels in cancer cell lines, and specific inhibitors of both PKC and IKK downregulate A3B expression. Using luciferase reporter and EMSA assays, we identify 3 NF-κΒ binding sites in the A3B promoter and reveal that NF-κB p65/p50 and p65/c-Rel heterodimers are important for A3B transcription. These results suggest that the classical NF-κB pathway is responsible for activation of A3B mRNA expression and further imply that inhibition of PKC and IKK might augment cancer treatment by reducing cancer progression and metastasis through downregulation of A3B expression.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminases have emerged as potential genomic mutators in various cancers. Multiple myeloma accumulates APOBEC signature mutations as it progresses; however, the mechanisms underlying APOBEC signature acquisition and its consequences remain elusive. In this study, we examined the significance and clinical impact of APOBEC3B (A3B) activity in multiple myeloma. Among APOBECs, only highly expressed A3B was associated with poor prognosis in myeloma patients, independent of other known poor prognostic factors. Quantitative PCR revealed that CD138-positive primary myeloma cells and myeloma cell lines exhibited remarkably high A3B expression levels. Interestingly, lentiviral A3B knockdown prevented the generation of deletion and loss-of-function mutations in exogenous DNA, whereas in control cells, these mutations accumulated with time. A3B knockdown also decreased the basal levels of γ-H2AX foci, suggesting that A3B promotes constitutive DNA double-strand breaks in myeloma cells. Importantly, among control shRNA-transduced cells, we observed the generation of clones that harboured diverse mutations in exogenous genes and several endogenous genes frequently mutated in myeloma, including TP53 . Taken together, the results suggest that A3B constitutively mutates the tumour genome beyond the protection of the DNA repair system, which may lead to clonal evolution and genomic instability in myeloma.

APOBEC3B cytidine deaminase (A3B) catalyzes cytosine into uracil in single-strand DNA and induces C-to-T mutations in genomic DNA of various types of tumors. Accumulation of APOBEC signature mutations is correlated with a worse prognosis for patients with breast cancer or multiple myeloma, suggesting that A3B activity might be a cause of the unfavorable DNA mutations and clonal evolution in these tumors. Phosphorylation of conserved threonine residues of other cytidine deaminases, activation induced deaminase (AID) and APOBEC3G, inhibits their activity. Here we show that protein kinase A (PKA) physically binds to A3B and phosphorylates Thr214. In vitro deaminase assays and foreign DNA editing assays in cells confirm that phosphomimetic A3B mutants, T214D and T214E, completely lose deaminase activity. Molecular dynamics simulation of A3B phosphorylation reveals that Thr214 phosphorylation disrupts binding between the phospho-A3B catalytic core and ssDNA. These mutants still inhibit retroviral infectivity at least partially, and also retain full anti-retrotransposition activity. These results imply that PKA-mediated phosphorylation inhibits A3B mutagenic activity without destructing its innate immune functions. Therefore, PKA activation could reduce further accumulation of mutations in A3B overexpressing tumors.

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Kindler syndrome, a distinct type of epidermolysis bullosa, is a rare disorder caused by mutations in FERMT1, encoding kindlin-1. Most FERMT1 mutations lead to premature termination codons and absence of kindlin-1. Here we investigated the molecular and cellular consequences of a naturally occurring FERMT1 mutation, c.299_301del resulting in a single amino acid deletion, p.R100del. The mutation led to a 50% reduction of FERMT1 mRNA and 90% reduction of kindlin-1 protein in keratinocytes derived from the patient, as compared with control cells. The misfolded p.R100del kindlin-1 mutant was lysosomally degraded and launched a homeostatic unfolded protein response. Sodium-phenylbutyrate significantly increased kindlin-1 mRNA and protein levels and the area of mutant cells, acting as a chemical chaperone and probably also as a histone deacetylase inhibitor. In a recombinant system, low levels of wild-type or p.R100del mutant kindlin-1 were sufficient to improve the cellular phenotype in respect of spreading and proliferation as compared with kindlin-1 negative keratinocytes. The study of this hypomorphic mutation provides evidence that low amounts of kindlin-1 are sufficient to improve the epidermal architecture and Kindler syndrome cellular phenotype and proposes a personalized chaperone therapy for the patient.