GMCL1 Controls 53BP1 Stability and Modulates Paclitaxel Sensitivity in Cancer

Reviewer #1 (Public review):

In this manuscript, Pagano and colleagues test the idea that the protein GMCL1 functions as a substrate receptor for a Cullin RING 3 E3 ubiquitin ligase (CUL3) complex. Using a pulldown approach, they identify GMCL1 binding proteins, including the DNA damage scaffolding protein 53BP1. They then focus on the idea that GMCL1 recruits 53BP1 for CUL3-dependent ubiquitination, triggering subsequent proteasomal degradation of ubiquitinated 53BP1.

In addition to its DNA damage signalling function, in mitosis, 53BP1 is reported to form a stopwatch complex with the deubiquitinating enzyme USP28 and the transcription factor p53 (PMID: 38547292). These 53BP1-stopwatch complexes generated in mitosis are inherited by G1 daughter cells and help promote p53-dependent cell cycle arrest independent from DNA damage (PMID: 38547292). Several studies show that knockout of 53BP1 overcomes G1 cell cycle arrest after mitotic delays caused by anti-mitotic drugs or centrosome ablation (PMID: 27432897, 27432896). In this model, it is crucial that 53BP1 remains stable in mitosis and more stopwatch complex is formed after delayed mitosis.

Pagano and coworkers suggest that 53BP1 levels can sometimes be suppressed in mitosis if the cells overexpress GMCL1. They carry out a bioinformatic analysis of available public data for p53 wild-type cancer cell lines resistant to the anti-mitotic drug paclitaxel and related compounds. Stratifying GMCL1 into low and high expression groups reveals a weak (p = 0.05 or ns) correlation with sensitivity to taxanes. It is unclear on what basis the authors claim paclitaxel-resistant and p53 wild-type cancer cell lines bypass the mitotic surveillance/timer pathway. They have not tested this. Figure 3 is a correlation assembled from public databases but has no experimental tests. Figure 4 looks at proliferation but not cell cycle progression or the length of mitosis. The main conclusions relating to cell cycle progression and specifically the link to mitotic delays are therefore not supported by experimental data. There is no imaging of the cell cycle or cell fate after mitotic delays, or analysis of where the cells arrest in the cell cycle. Most of the cell lines used have been reported to lack a functional mitotic surveillance pathway in the recent work by Meitinger. To support these conclusions, the stability of endogenous 53BP1 under different conditions in cells known to have a functional mitotic surveillance pathway needs to be examined. A key suggestion in the work is that the level of GMCL1 expression correlates with resistance to taxanes. For the mitotic surveillance pathway, the type of drug (nocodazole, taxol, etc) used to induce a delay isn't thought to be relevant, only the length of the delay. Do GMCL1-overexpressing cells show resistance to anti-mitotics in general?

Importantly, if GMCL1 specifically degrades 53BP1 during prolonged mitotic arrests, the authors should show what happens during normal cell divisions without any delays or drug treatments. How much 53BP1 is destroyed in mitosis under those conditions? Does 53BP1 destruction depend on the length of mitosis, drug treatment, or does 53BP1 get degraded every mitosis regardless of length? Testing the contribution of key mitotic E3 ligase activities on mitotic 53BP1 stability, such as the anaphase-promoting complex/cyclosome (APC/C) is important in this regard. One previous study reported an analysis of putative APC/C KEN-box degron motifs in 53BP1 and concluded these play a role in 53BP1 stability in anaphase (PMID: 28228263).

There is no direct test of the proposed mechanism, and it is therefore unclear if 53BP1 is ubiquitinated by a GMCL1-CUL3 ligase in cells, and how efficient this process would be at different cell cycle stages. A key issue is the lack of experimental data explaining why the proposed mechanism would be restricted to mitosis. Indirect effects, such as loss of 53BP1 from the chromatin fraction during M phase upon GMCL1 overexpression, do not necessarily mean that 53BP1 is degraded. PLK1-dependent chromatin-cytoplasmic shuttling of 53BP1 during mitotic delays has been described previously (PMID: 38547292, 37888778). These papers are cited in the text, but the main conclusions of those papers on 53BP1 incorporation into a stopwatch complex during mitotic delays have been ignored. Are the authors sure that 53BP1 is destroyed in mitosis and not simply re-localised between chromatin and non-chromatin fractions? At the very least, these reported findings should be discussed in the text.

The authors use a variety of cancer cell line models throughout their study, most of which have been reported to lack a functional mitotic surveillance pathway. U2OS and HCT116 cells do not respond normally to mitotic delays, despite being annotated as p53 WT. Other studies have used p53 wild-type hTERT RPE-1 cells to study the mitotic surveillance pathway. If the model is correct, then over-expressing GMCL1 in hTERT-RPE1 cells should suppress cell cycle arrest after mitotic delays, and GMCL1 KO should make the cells more sensitive to delays. These experiments are needed to provide an adequate test of the proposed model.

To conclude, while the authors propose a potentially interesting model on how GMCL1 overexpression could regulate 53BP1 stability to limit p53-dependent cell cycle arrest, it is unclear what triggers this pathway or when it is relevant. 53BP1 is known to function in DNA damage signalling, and GMCL1 might be relevant in that context. The manuscript contains the initial description of GMCL1-53BP1 interaction but lacks a proper analysis of the function of this interaction and is therefore a preliminary report.

Reviewer #2 (Public review):

Summary:

This study investigates the role of GMCL1 in regulating the mitotic surveillance pathway (MSP), a protective mechanism that activates p53 following prolonged mitosis. The authors identify a physical interaction between 53BP1 and GMCL1, but not with GMCL2. They propose that the ubiquitin ligase complex CRL3-GMCL1 targets 53BP1 for degradation during mitosis, thereby preventing the formation of the "mitotic stopwatch" complex (53BP1-USP28-p53) and subsequent p53 activation. The authors show that high GMCL1 expression correlates with resistance to paclitaxel in cancer cell lines that express wild-type p53. Importantly, loss of GMCL1 restores paclitaxel sensitivity in these cells, but not in p53-deficient lines. They propose that GMCL1 overexpression enables cancer cells to bypass MSP-mediated p53 activation, promoting survival despite mitotic stress. Targeting GMCL1 may thus represent a therapeutic strategy to re-sensitize resistant tumors to taxane-based chemotherapy.

Strengths:

This manuscript presents potentially interesting observations. The major strength of this article is the identification of GMCL1 as a 53BP1 interaction partner. The authors identified relevant domains and showed that GMCL1 controls 53BP1 stability. The authors further show a potentially interesting link between GMCL1 status and sensitivity to Taxol.

Weaknesses:

However, the manuscript is significantly weakened by unsubstantiated mechanistic claims, overreliance on a non-functional model system (U2OS), and overinterpretation of correlative data. To support the conclusions of the manuscript, the authors must show that the GMCL1-dependent sensitivity to Taxol depends on the mitotic surveillance pathway.

Reviewer #3 (Public review):

Summary:

In this study, Kito et al follow up on previous work that identified Drosophila GCL as a mitotic substrate recognition subunit of a CUL3-RING ubiquitin ligase (CRL3) complex.

Here they characterize mutants of the human ortholog of GCL, GMCL1, that disrupt the interaction with CUL3 (GMCL1E142K) and that lack the substrate interaction domain (GMCL1 BBO). Immunoprecipitation followed by mass spectrometry identified 9 proteins that interacted with wild-type FLAG-GMCL1 and GMCL1 EK but not GMCL1 BBO. These proteins included 53BP1, which plays a well-characterized role in double-strand break repair but also functions in a USP28-p53-53BP1 "mitotic stopwatch" complex that arrests the cell cycle after a substantially prolonged mitosis. Consistent with the IP-MS results, FLAG-GMCL1 immunoprecipitated 53BP1. Depletion of GMCL1 during mitotic arrest increased protein levels of 53BP1, and this could be rescued by wild-type GMCL1 but not the E142K mutant or a R433A mutant that failed to immunoprecipitate 53BP1.

Using a publicly available dataset, the authors identified a relatively small subset of cell lines with high levels of GMCL1 mRNA that were resistant to the taxanes paclitaxel, cabazitaxel, and docetaxel. This type of analysis is confounded by the fact that paclitaxel and other microtubule poisons accumulate to substantially different levels in various cell lines (DOI: 10.1073/pnas.90.20.9552 , DOI: 10.1091/mbc.10.4.947 ), so careful follow-up experiments are required to validate results. The correlation between increased GMCL1 mRNA and taxane resistance was not observed in lung cancer cell lines. The authors propose this was because nearly half of lung cancers harbor p53 mutations, and lung cancer cell lines with wild-type but not mutant p53 showed the correlation between increased GMCL1 mRNA and taxane resistance. However, the other cancer cell types in which they report increased GMCL1 expression correlates with taxane sensitivity also have high rates of p53 mutation. Furthermore, p53 status does not predict taxane response in patients (DOI: 10.1002/1097-0142(20000815)89:4<769::aid-cncr8>3.0.co;2-6 , DOI: 10.1002/(SICI)1097-0142(19960915)78:6<1203::AID-CNCR6>3.0.CO;2-A , PMID: 10955790).

The authors then depleted GMCL1 and reported that it increased apoptosis in two cell lines with wild-type p53 (MCF7 and U2OS) due to activation of the mitotic stopwatch. This is surprising because the mitotic stopwatch paper they cite (DOI: 10.1126/science.add9528 ) reported that U2OS cells have an inactive stopwatch and that activation of the stopwatch results in cell cycle arrest rather than apoptosis in most cell types, including MCF7. Beyond this, it has recently been shown that the level of taxanes and other microtubule poisons achieved in patient tumors is too low to induce mitotic arrest (DOI: 10.1126/scitranslmed.3007965 , DOI: 10.1126/scitranslmed.abd4811 , DOI: 10.1371/journal.pbio.3002339 ), raising concerns about the relevance of prolonged mitosis to paclitaxel response in cancer. The findings here demonstrating that GMCL1 mediates degradation of 53BP1 during mitotic arrest are solid and of interest to cell biologists, but it is unclear that these findings are relevant to paclitaxel response in patients.

Strengths:

This study identified 53BP1 as a target of CRL3GMCL1-mediated degradation during mitotic arrest. AlphaFold3 predictions of the binding interface, followed by mutational analysis, identified mutants of each protein (GMCL1 R433A and 53BP1 IEDI1422-1425AAAA) that disrupted their interaction. Knock-in of a FLAG tag into the C-terminus of GMCL1 in HCT116 cells, followed by FLAG immunoprecipitation, confirmed that endogenous GMCL1 interacts with endogenous CUL3 and 53BP1 during mitotic arrest.

Weaknesses:

The clinical relevance of the study is overinterpreted. The authors have not taken relevant data about the clinical mechanism of taxanes into account. Supraphysiologic doses of microtubule poisons cause mitotic arrest and can activate the mitotic stopwatch. However, in physiologic concentrations of clinically useful microtubule poisons, cells proceed through mitosis and divide their chromosomes on mitotic spindles that are at least transiently multipolar. Though these low concentrations may result in a brief mitotic delay, it is substantially shorter than the arrest caused by high concentrations of microtubule poisons, and the one mimicked here by 16 hours of 0.4 mg/mL nocodazole, which is not used clinically and does not induce multipolar spindles. Resistance to mitotic arrest occurs through different mechanisms than resistance to multipolar spindles. No evidence is presented in the current version of the manuscript that GMCL1 affects cellular response to clinically relevant doses of paclitaxel.