APOBEC3B Induction Following DNA Damage Response Modulates the Survival and Treatment Response in Human Multiple Myeloma
Authors: Xing Lijie , Liu Jiye , Li Yuyin , Lin Liang , Wen Kenneth , Cho Shih-Feng , Yu Tengteng , An Gang , Qiu Lugui , Munshi Nikhil C. , Anderson Kenneth C. , Tai Yu-Tzu 
Published: November 2019

Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B, A3B) is one of 7-membered DNA cytosine deaminase family, causing cytosine-to-uracil (C-to-U) deamination in single-stranded DNA and promoting mutations in multiple human cancers including multiple myeloma (MM). High APOBEC3B expression is found in a significant portion of MM patients with MAF overexpression among t(14;16) and t(14;20). A3B upregulation is further associated with poor prognosis in MM, suggesting its role in the MM pathophysiology. However, approximately 23% MM patients with high APOBEC3 activity are associated with MAF/MAFA/MAFB translocations, the remainder of patients with high APOBEC3 carry neither translocations nor overexpression of these genes. Besides, studies are lacking on how A3B is regulated and the role of A3B in drug responses in MM. We here defined new mechanisms controlling A3B expression and further characterized its impact on treatment responses to current anti-MM therapies. Using qRT-PCR, A3B transcript is significantly higher than other members of the APOBEC3 gene family in MM cell lines (n=19) and MM patients, indicating that A3B may play a major role in MM. Using immunoblotting analysis, A3B protein expression was further confirmed in MM cell lines with various levels (n=10). Importantly, A3B mRNA upregulation by 1.34-42.64 folds was observed in CD138-purified cells from majorities of MM patients (83.3%) when compared to PBMC from the same individual (n=12). In MM cell lines without MAF/MAFA/MAFB translocation as a study model, higher A3B protein expression is associated with higher DNA damage levels as evidenced by higher γ-H2AX. These results suggest that A3B expression might be influenced by DNA damage levels in MM cells. Following a short time treatment of gamma-irradiation to cause DNA damages, A3B expression in viable MM cells was enhanced in a dose-dependent manner. We next treated MM cells (n=5) with common anti-MM drugs such as Melphalan (Mel) and Bortizomib (btz), both of which induce DNA damages, followed by examination of changes in A3B and γ-H2AX. Under sublethal treatment conditions of Mel or btz, A3B was consistently induced at both mRNA and protein levels in multiple MM cell lines regardless of the baseline A3B expression. Significantly, A3B was upregulated and associated with increased γ-H2AX in patient MM cells treated with Mel or btz under sub-lethal doses. Since DNA damages activate the ATR/ATM pathway, we next investigated whether these kinases mediate A3B induction following treatments with these compounds in MM cells. The presence of ATM or ATR inhibitors blocked A3B upregulated by these DNA damage-inducing treatments in MM cell lines (n=3), indicating an ATM/ATR-dependent pathway for A3B changes. Next, gene-specific CRISPR knock out (KO) and inducible-shRNA knockdown (KD) were used to determine the functional impact of perturbation of A3B in proliferation and survival of MM cells. Both KO and KD of A3B decreased growth and viability of MM cell lines regardless of sensitive or resistant to dexamethasone or lenalidomide. Using LIVE/DEAD fixable Aqua Stain and annexin V-based flow cytometric analysis, A3B inhibition enhanced growth arrest followed by apoptosis in MM cells. Significantly, A3B KD by its shRNA in RPMI8226 MM cells enhanced sensitivity to pomalidomide. Taken together, these data indicate that increased A3B level plays a critical role in MM cell survival and drug responses. DNA damages triggered by IR, Mel, or btz further enhance A3B expression via ATM/ATR pathway, which in turn increases subclonal diversity leading to drug resistance. The role of A3B in disease pathophysiology and progression, coupled with its function in mediating treatment response, suggest potential utility of targeting A3B in MM.

Disclosures
Munshi: Celgene: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Takeda: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.