RAD54L2 counters TOP2-DNA adducts to promote genome stability

Abstract

The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.

Fig. 1.. Focused CRISPR-Cas9 screens identify factors whose loss imparts etoposide hypersensitivity.

(A) Layout of CRISPR-Cas9 screens in WT and TP53^KO RPE-1 cells [multiplicity of infection (MOI), drug concentration reducing cell viability by 50% (IC₅₀)]. Figure generated with BioRender. (B) Biplot showing gene enrichment scores (NormZ) upon etoposide treatment in WT (x axis) and TP53^KO (y axis) RPE-1 cells. Blue or orange circles indicate known or previously unknown factors, respectively, to affect etoposide sensitivity. (C to G) MTT cell viability assays of two HAP1 RAD54L2^KO clones upon treatment with etoposide (C), camptothecin (D), ionizing radiation (E), ICRF-193 (F), or hydroxyurea (G). n = 3 independent experiments. Bars represent means ± SEM.

Fig. 2.. RAD54L2 binds SUMO and TOP2α/β and requires its SIMs to interact with SUMO-modified TOP2α/β.

(A and B) Volcano plots showing RAD54L2 interactors identified by IP-MS in untreated (UT) cells (A) or etoposide (ETP)–treated cells. (B) Green dots bordered by dotted lines indicating P values ≤0.05 and log₂ fold changes ≥2 are significant interactors. (C) Schematic of the putative domains and relevant properties of RAD54L2. The K311A catalytic site mutation is labeled, and the SIMs are highlighted in blue dots. (D) Clonogenic survival assays of control (CTRL) cells or RAD54L2^KO cells complemented with vectors expressing mCherry, mCherry-RAD54L2^WT, mCherry-RAD54L2^K311A mutant, or mCherry-RAD54L2^ΔSIM mutant; n = 3 independent experiments. Bars represent means ± SEM. (E) Coimmunoprecipitation (IP) of mCherry from extracts of untreated or etoposide treated RPE-1 cells stably expressing mCherry (EV), mCherry-RAD54L2^WT, or mCherry-RAD54L2^ΔSIM mutant. This experiment was repeated six times with similar results.

Fig. 3.. RAD54L2 operates through a mechanism dependent on ZNF451 and independent of TDP2.

(A) Biplot showing gene enrichment scores (NormZ) upon etoposide treatment in TP53^KO (x axis) and TP53/RAD54L2^KO (y axis) RPE-1 cells. Orange dots relate to well-characterized DDR factors, most discussed in the text. (B) Clonogenic survival assays of CTRL, RAD54L2^KO, TDP2^KO, and TDP2/RAD54L2^KO RPE-1 cells upon treatment with etoposide. n = 3 independent experiments. Bars represent means ± SEM. Competitive growth assays of RPE-1 CTRL (C), LIG41^KO (D), BRCA1^KO (E), and ZNF451^KO (G) cells transduced with virus expressing the indicated sgRNAs in the presence of various etoposide concentrations; n = 3 independent experiments. Bars represent means ± SEM. (F) Clonogenic survival assays of CTRL or ZNF451^KO transduced with a CTRL sgRNA or with an sgRNA targeting RAD54L2. n = 3 independent experiments. Bars represent means ± SEM. (H) Extracts of etoposide-treated CTRL or ZNF451^KO RPE-1 cells stably expressing mCherry [empty vector (EV)] or mCherry-RAD54L2^WT were subjected to mCherry IP followed by Western blotting for the indicated proteins. This experiment was repeated three times with similar results.

Fig. 4.. RAD54L2 counters TOP2ccs by promoting their resolution.

(A) Quantification of γH2AX foci in G₁ cells (cyclin A negative). Cells were treated with 30 μM etoposide for 30 min, washed, and left to recover for 4 or 8 hours. n > 3 independent experiments. Bars represent means ± SEM. One-way analysis of variance (ANOVA) was used for statistical testing. (B) DUST assays in CTRL or RAD54L2^KO HAP1 cells collected after 1 hour of treatment with 20 μM etoposide or 30 min or 1 hour after etoposide washout. This experiment was carried out three times with similar results. (C) Quantifications of TOP2α or TOP2β signal from the DUST assays. Bars represent means ± SEM. (D) Proposed mechanism for RAD54L2 in TOP2cc resolution. UBC9, SUMO conjugating enzyme 2 ligase. Figure generated with BioRender. ns, not significant.