Cyclin D1 overexpression induces replication stress and microhomology-mediated end-joining dependence in mantle cell lymphoma
Jithma P. Abeykoon, Shuhei Asada, Guangli Zhu, Yuna Hirohashi, Lisa Moreau, Divya Iyer, Sirisha Mukkavalli, Kalindi Parmar, Gabriella Zambrano, Lige Jiang, Dongni Yi, Michelle Manske, Kimberly Gwin, Rebecca L. King, James R. Cerhan, Xiaosheng Wu, Zhenkun Lou, Geoffrey I. Shapiro, Thomas Witzig, Alan D’Andrea
Jithma P. Abeykoon, Shuhei Asada, Guangli Zhu, Yuna Hirohashi, Lisa Moreau, Divya Iyer, Sirisha Mukkavalli, Kalindi Parmar, Gabriella Zambrano, Lige Jiang, Dongni Yi, Michelle Manske, Kimberly Gwin, Rebecca L. King, James R. Cerhan, Xiaosheng Wu, Zhenkun Lou, Geoffrey I. Shapiro, Thomas Witzig, Alan D’Andrea
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Research Article Cell biology Hematology

Cyclin D1 overexpression induces replication stress and microhomology-mediated end-joining dependence in mantle cell lymphoma

Abstract

Oncogene expression can cause replication stress (RS), leading to DNA double-strand breaks (DSBs) that require repair through pathways such as homologous recombination, nonhomologous end-joining, and microhomology-mediated end-joining (MMEJ). Cyclin D1 (encoded by CCND1) is a well-known oncoprotein overexpressed in cancer; however, its role in RS is unknown. Using mantle cell lymphoma (MCL) as a naturally occurring model of cyclin D1 overexpression, we examined the impact of cyclin D1 on RS and DSB repair mechanisms. Cyclin D1 overexpression elevated RS, increased DNA damage, especially during mitosis, and caused specific upregulation of MMEJ. Furthermore, cyclin D1 activated polymerase theta (POLQ) transcription by binding its promoter loci, driving POLΘ-mediated MMEJ that is essential to withstand cyclin D1–induced RS. Moreover, concurrent ATM deficiency further intensified RS, enhanced POLQ expression, and heightened reliance on MMEJ-mediated DNA damage repair. Consequently, inhibition of POLΘ in cyclin D1–overexpressed settings further exacerbated RS, causing single-strand DNA gap accumulations and chromosomal instability, ultimately leading to apoptosis, an effect amplified in ATM-deficient cells. Targeting MMEJ via POLΘ inhibition is therefore an effective strategy in the context of cyclin D1 overexpression and ATM deficiency and may provide a unique therapeutic approach for treating MCL and other malignancies characterized by similar alterations.

Authors

Jithma P. Abeykoon, Shuhei Asada, Guangli Zhu, Yuna Hirohashi, Lisa Moreau, Divya Iyer, Sirisha Mukkavalli, Kalindi Parmar, Gabriella Zambrano, Lige Jiang, Dongni Yi, Michelle Manske, Kimberly Gwin, Rebecca L. King, James R. Cerhan, Xiaosheng Wu, Zhenkun Lou, Geoffrey I. Shapiro, Thomas Witzig, Alan D’Andrea

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Figure 3

Cyclin D1 overexpression specifically increases the expression of POLQ through binding to the POLQ promoter, which leads to increased MMEJ-mediated DNA damage repair.

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Cyclin D1 overexpression specifically increases the expression of POLQ t...
(A) Assessment of POLQ expression and DNA damage (γ-H2AX) in MCL cells (Jeko) when cyclin D1 expression is decreased through CRISPRi technology. (B) Quantification of POLQ and CCND1 transcription in Jeko cells with decreased cyclin D1 expression (experiments were done in quadruplicates, and P value was calculated using t test). (C) Assessment of MMEJ-mediated DNA damage repair in Jeko cells with decreased cyclin D1 expression (experiments were done in quadruplicates, and P value was calculated using t test). (D) Assessment of sensitivity to POLΘ inhibition (ART558) in Jeko cells with decreased cyclin D1 expression (experiments were done in triplicates, and P value was calculated using t test). (E) Assessment of POLQ expression with lentiviral-mediated cyclin D1 overexpression in multiple NHL cell lines belonging to T cell lymphoma (SR786 and Karpas 299) and diffuse large B cell lymphoma (Ly1, Ly19, and DHL6) (experiments were done in triplicates, and P value was calculated using t test). (F) Evaluation of cyclin D1 binding to the POLQ promoter region through ChIP in HA-tagged cyclin D1–overexpressing MCL cells (Jeko cells with endogenous cyclin D1 expression stably decreased) (experiments were done in triplicates, and P value was calculated using 1-way ANOVA with Tukey’s post hoc test). (G) Assessment of the transcriptional activity of the POLQ promoter using a luciferase assay in cells with cyclin D1 overexpression (experiments were done in triplicates, and P value was calculated using 1-way ANOVA with Tukey’s post hoc test). Fluc, Firefly luciferase; Rluc, Renilla luciferase; OE, overexpressed. *P < 0.05; **P < 0.01; ***P < 0.001. Data are shown as the mean ± SEM.