DNA Polymerases Pol θ/Pol η Involved in Error-Prone DNA Repair Are Highly Expressed in Multiple Myeloma and Upregulated By DNA Damage

Background: DNA polymerases (DNA pols) are essential enzymes for DNA replication. In mammalian cells, DNA pols are divided into four families: A (Pol θ, Pol γ, and Pol ν), B (Pol α, Pol δ, Pol ε, and Pol ζ), X (Pol β, Pol λ, Pol μ, and TDT), and Y (Pol η, Pol ι, Pol κ, and REV1). These DNA pols are required for both genome duplication and protecting cells from DNA damage induced by endogenous and exogenous agents, such as ROS, UV, and chemotherapeutic drugs. For example, Pol β, Pol λ, and Pol ι participate in base excision repair. Contrastingly, Pol ζ, REV1, Pol η, Pol ι, and Pol κ can replicate over various DNA lesions to prevent DNA replication stalling, known as translesion synthesis. Although some DNA pols are highly expressed in cancer cells, indicating chemotherapeutic resistance and poor outcome, their exact roles and expression mechanisms have not been fully elucidated. Multiple myeloma (MM) is a hematological malignancy of terminally differentiated plasma cells, with multistep progression from pre-cancer stage namely. In this study we attempted to elucidate the involvement of DNA pols in multistep oncogenesis of MM.

Methods: A total of 63 MM and 29 MGUS patients, 15 controls, and 9 MM cell lines were included in the study. RNA was extracted from purified CD138+ plasma cells. DNA pol expressions were determined by RQ-PCR. Their expression levels were normalized against ACTB levels and calculated with 2^-ΔΔCt value. Doxycycline-inducible p53 system (Tet-on p53) and nutlin-3 were used for analyzing the role of p53 in DNA pol expressions in MM cell lines. Melphalan, doxorubicin, and bortezomib were used to examine DNA pol expressions in damaged cells in vitro. JQ1 and CPI203 were used to evaluate the role of bromodomain in DNA pol expressions.

Results: Pol α and Pol ε expressions were significantly higher in MM than in control (p=0.007 and p=0.004, respectively), but Pol ε and Pol ζ levels were not significantly different (p=0.631, p=0.0826, respectively). Pol η, REV1, Pol ι, and Pol κ expressions were significantly higher in MM than control (p<0.001, p=0.002, p<0.001, and p<0.001, respectively). Pol θ and Pol γ were expressed at a higher level in MM than in control (p<0.001 and p<0.001, respectively). Pol β and Pol λ expressions were higher in MM than in control (p=0.0088 and p=0.013, respectively). Although the expressions of many DNA pols were higher in MM plasma cells, we focused on Pol η and Pol θ, because Pol λ, Pol μ, Pol ν, and Pol ι were expressed at very low levels, and Pol ε, Pol ζ, Pol γ, Pol κ, and REV1 were expressed in PBMNCs of healthy volunteers at high level. Pol η and Pol θ expressions did not differ due to known risk factors, such as cytogenetic abnormalities and ISS. Pol η expressions were positively correlated with p53 and myc expressions (r=0.718, p<0.001, r=0.528, p<0.001 respectively). p53 overexpression by Tet-on vector or nutlin-3 treatment enhanced Pol η expression, indicating that Pol η expression is regulated by p53. Melphalan or doxorubicin increased Pol η expression, but bortezomib or lenalidomide did not, suggesting that Pol η is upregulated by DNA damage via p53 pathway. Overall survival of the patients with high Pol η expression tended to be worse than with low Pol η expression (24 months survival: 69.6% vs. 57.9%, p=0.29). Pol θ expression was weakly correlated with p53. Melphalan induced Pol θ expression but doxorubicin did not. JQ1 significantly reduced Pol θ expression suggesting that Pol θ was regulated by bromodomain.

Conclusion: We found that Pol θ and Pol η are highly expressed in MM, and upregulated by DNA damage. These DNA pols are involved in drug resistance and genomic instability leading to poor prognosis. Thus, DNA pols can be used as novel therapeutic targets and prognostic markers.