Introduction

Mitosis is orchestrated by several intracellular signaling pathways to ensure proper cell division while maintaining genomic integrity. Errors during cell division, including chromosome mis-segregation or spindle defects, could lead to changes in chromosome content, producing aneuploid or polyploid progeny cells, which could be detrimental during development or lead to oncogenesis^1,2. Therefore, cells have evolved quality control mechanisms to ensure proper cell division during M phase. One such surveillance mechanism is known as the Mitotic Surveillance Pathway (MSP), which senses prolonged mitosis as an indicator of mitotic defects³.

If a threshold mitotic duration is surpassed, the MSP induces cell cycle arrest in the daughter cells in a p53-dependent manner^4–6. During abnormally prolonged mitosis^7–10, the MSP is triggered by the formation of a ternary “mitotic stopwatch” complex consisting of the deubiquitinase USP28 (ubiquitin-specific protease 28), 53BP1 (p53-binding protein 1), which acts as a substrate recognition subunit, and p53. During delayed mitosis, formation of the mitotic stopwatch complex allows USP28 to deubiquitinate and subsequently protect p53 from MDM2-mediated degradation. The 53BP1-USP28-p53 complex accumulates and persists in the G1 phase of daughter cells, where it promotes cell cycle arrest via p53-mediated upregulation of p21 expression.

Notably, proper mitotic arrest plays a crucial role in the mechanism of microtubule-targeting therapies, such as taxanes (e.g., paclitaxel and docetaxel), which disrupt spindle formation and chromosomal segregation. Recent studies have shown that pharmacological interventions targeting M phase, even without resulting in complete mitotic arrest, can induce controlled cell death¹¹. For example, low dose paclitaxel treatments results in increased p21 expression and apoptosis in daughter cells¹². However, there are cancers that display resistance or insensitivity to treatment with taxanes, including metastatic breast cancer, ovarian cancer, and non-small cell lung cancer^13–15. Therefore, there is an urgent need to elucidate the strategies of paclitaxel resistance in such cancers.

Human germ cell-less protein-like 1 (GMCL1) is a putative substrate receptor of one of many CUL3-RING ubiquitin ligases (CRL3s). However, to date, GMCL1’s biological role and its substrates have remained uncharacterized. GMCL1 received its namesake from its Drosophila melanogaster homolog, GCL (Germ Cell-Less), which plays essential roles in early embryonic development and germ cell determination^16,17. GMCL1’s role in germ cell development appears to have been evolutionarily conserved as loss of GMCL1 expression in mice has been shown to cause defects in meiosis and spermiogenesis¹⁸, and altered GMCL1 expression was functionally associated with human asthenozoospermia¹⁹. Although GMCL1 homologs have been primarily associated with germ cell biology, several databases (e.g., GTEx²⁰ and GENT2²¹) indicate that GMCL1 is expressed in somatic cells as well, suggesting broader biological functions beyond germ cell development. In contrast, the GMCL1 paralog GMCL2 is specifically expressed in germ cells.

Here, we present studies that shed light on the role of GMCL1 in somatic cells. We demonstrate that, like its D. melanogaster counterpart, GMCL1 acts as a CRL3 substrate receptor. We also identify 53BP1 as a bona fide substrate of CRL3^GMCL1, demonstrating that its levels are regulated by GMCL1 during M phase. Reduction of mitotic 53BP1 by GMCL1 prevents the formation of the USP28-p53-53BP1 mitotic stopwatch complex and reduces p53 transmission to daughter cells. We therefore propose that GMCL1 inhibition, in combination with paclitaxel therapy, offers a promising strategy to overcome taxane resistance.

Results

Identification of 53BP1 as an interactor of CRL3^GMCL1

We have shown that the GMCL1 ortholog in D. melanogaster, GCL, is a substrate recognition subunit of a CRL3 complex that is active specifically in mitosis²². So, we predicted the human GMCL1 to also behave as a CRL3 substrate receptor. GMCL1 contains a BTB (Broad-Complex, Tramtrack, and Bric-a -brac) and a BACK (BTB and C-terminal Kelch) domain (Supplementary Figure 1A), consistent with other CRL3 receptors. On its C-terminus, GMCL1 also contains a GCL domain (residues 379-515), which is distinct from the Kelch domains commonly used by other CUL3 substrate receptors since it is predicted to form a β-sandwich characterized by two opposing anti-parallel β-sheets, each made up of four β-strands^22,23 (Supplementary Figure 1A). A Dali search²⁴ of GMCL1’s C-terminal domain reveals that it has some structural homology to the MATH (meprin and TRAF homology) domain used by another CRL3 substrate receptor, SPOP²⁵, suggesting that the predicted structure of GMCL1’s GCL domain could also potentially act as a protein-protein interaction motif to recruit substrates.

To investigate the role of GMCL1 in somatic cells, we used immunoprecipitation followed by mass spectrometry (IP-MS) to identify binding partners of GMCL1. Proteomics studies were performed by expressing and purifying the following FLAG-tagged proteins: (i) wild-type GMCL1 (GMCL1 WT), (ii) GMCL1 E142K (GMCL1 EK), which carries a mutation in the BTB domain that is predicted to disrupt its interaction with CUL3, and (iii) GMCL1 BTB/BACK-only (GMCL1 BBO) that lacks the GCL domain (Figure 1A-C). IP-MS analysis identified 1,765 potential binding partners that specifically interact with GMCL1 via its C-terminal domain. Using SAINT scores > 0.70 and FDR < 5%, this list was refined to 9 proteins that showed significant interaction with GMCL1 WT and GMCL1 EK, but not with GMCL1 BBO, with 53BP1 being the one of most enriched protein (Figure 1B, Supplementary Table 1). This selective enrichment suggests that the C-terminal, “MATH-like” GCL domain of GMCL1 is critical for its interaction with binding partners, including 53BP1. The interaction between GMCL1 and endogenous 53BP1 and CUL3 was validated using immunoprecipitations (IPs) followed by immunoblotting (Figure 1C,D).

Figure 1.

To further study the direct interaction between GMCL1 and 53BP1, we used AlphaFold 3²⁶ to predict their binding interface and identify potential critical residues at the recognition interface between the substrate receptor and 53BP1to see if mutating these residues could abolish their interaction. Consistent with our initial IP-MS experiment, the Alphafold 3 model predicted that the C-terminal domain of GMCL1 would interact with 53BP1. Based on the GMCL1-53BP1 complex structure prediction, we identified Arg 433, which appears to be a solvent-exposed residue in GMCL1’s GCL domain that could interact with 53BP1 without impeding GMCL1’s binding to with CUL3. Thus, we generated the GMCL1 R433A (GMCL1 RA) point mutant and tested its binding to 53BP1 using IP. As predicted, compared to WT GMCL1, the R433A mutation completely abolished the binding of GMCL1 to 53BP1, but did not impact GMCL1’s binding to CUL3 (Figure 1D).

To determine which region of 53BP1 mediates its binding to GMCL1, we mapped the predicted GMCL1-binding site on 53BP1 and identified a conserved IEDI amino acid sequence within the Minimal Focus Forming region (MFF) of 53BP1²⁷ (Supplementary Figure 1B-E). Through IPs, we demonstrate that a 53BP1 mutant either lacking the MFF region or containing the IEDI-to-AAAA mutation within this region lost its interaction with CRL3^GMCL1, suggesting that this conserved IEDI sequence within 53BP1’s MFF forms a critical degron recognized by GMCL1 (Figure 1E).

To examine the interaction between endogenous GMCL1 and endogenous 53BP1, we used CRISPR-Cas9 to knock in a FLAG tag at the C-terminus of GMCL1. Immunoprecipitation experiments confirmed that endogenous GMCL1 interacts with both endogenous CUL3 and 53BP1 (Figure 1F).

Finally, we sought to determine whether GMCL2, a GMCL1 paralog, is also able to interact with 53BP1. Immunoprecipitation of FLAG-tagged GMCL1 or GMCL2 from HEK293T cells revealed that while GMCL1 binds to 53BP1, GMCL2 does not (Supplementary Figure 1F), suggesting that GMCL1 and GMCL2 have distinct functions.

Overall, our results suggest that GMCL1 is a bona fide CRL3 substrate receptor and interacts specifically with 53BP1.

GMCL1 loss leads to 53BP1 accumulations under prolonged M phase

To investigate whether GMCL1 regulates 53BP1 stability, we generated GMCL1 knock-out (KO) cells using CRISPR Cas-9²⁸ and compared 53BP1 levels between GMCL1 WT and two GMCL1 KO clones. We found that 53BP1 levels were significantly increased in our GMCL1 KO cells during M phase. While the whole cell extracts (WCE) showed modest differences in GMCL1 levels between the GMCL1 WT and KO clones, our fractionation experiments revealed that 53BP1 mainly accumulated in the chromatin-bound fraction of GMCL1 KO cells (Figure 2A).

Figure 2.

To further explore the role of GMCL1 on 53BP1 stability, we stably reconstituted U2OS GMCL1 KO cells with either GMCL1 WT or binding mutants (i.e., GMCL1 EK and GMCL1 RA). Notably, the accumulation of 53BP1 in GMCL1 KO cells was rescued only upon re-expression of GMCL1 WT. In contrast, 53BP1 levels remained high in GMCL1 KO cells expressing either GMCL1 EK or GMCL1 RA (Figure 2B), emphasizing the importance of GMCL1’s ability to bind both CUL3 (through the E142 residue) and 53BP1 (through the R433 residue) to regulate 53BP1 levels. Furthermore, GMCL1 WT re-expression in GMCL1 KO cells led to decreased mRNA levels of p21 and NOXA during mitosis, whereas the GMCL1 EK and GMCL1 RA mutants failed to suppress these transcripts (Supplementary Figure 2A).

Collectively, these findings indicate that GMCL1 modulates 53BP1 availability through interactions with the CRL3^GMCL1 complex, playing a critical role in the regulation of 53BP1 levels in M phase.

During mitosis, 53BP1 has been implicated in monitoring centrosome integrity and mitotic progression via the Mitotic Stopwatch Pathway (MSP). A recent study demonstrated that cells experiencing abnormally prolonged mitosis transmit this information to daughter cells though a ternary complex containing 53BP1, USP28, and p53 called the “mitotic stopwatch” complex⁷.

To investigate whether GMCL1 regulates this complex, we analyzed GMCL1 levels in post-mitotic daughter cells seven hours after release from prolonged nocodazole arrest. GMCL1 KO daughter cells, reconstituted with GMCL1 WT, successfully re-entered the cell cycle, exhibiting low levels of 53BP1, p53, and p21, along with reduced expression of apoptosis-related genes (Supplementary Figure 2B,C). In contrast, cells reconstituted with either the E142K or R433A mutant displayed persistently elevated levels of 53BP1, p53, and p21, accompanied by increased expression of apoptosis-related genes (Supplementary Figure 2B,C). Notably, compared to parental cells, GMCL1 KO cells, whether reconstituted with WT or mutant GMCL1 constructs, showed no significant alterations in cell cycle profiles (Supplementary Figure 2D).

Next, we used cycloheximide (CHX) to inhibit translation and to assess the stability of 53BP1. We observed that in the chromatin-bound fraction of GMCL1 KO cells, 53BP1 was more stable compared to cells rescued with GMCL1 WT (Figure 2C).

These results suggest that cells lacking GMCL1 that experienced prolonged mitosis will maintain elevated levels of p53, increasing the likelihood of apoptosis in subsequent cell cycles.

GMCL1 modulates paclitaxel resistance in cancer cells

Paclitaxel, a widely used chemotherapeutic drug, stabilizes microtubules and prevents their depolymerization, thereby halting cancer cell division and inducing apoptosis. To assess the clinical significance of GMCL1, we investigated the relationship between GMCL1 expression levels and Taxol resistance in various cancer cell lines. To this end, we leveraged the PRISM (profiling relative inhibition simultaneously in mixtures) repurposing dataset, which compiles the proliferation inhibitory activity of 4,518 compounds tested across 578 patient-derived cancer cell lines²⁹. This high-throughput screening dataset provides valuable insights into drug resistance mechanisms. We examined the impact of GMCL1 mRNA expression on Taxol resistance by analyzing taxane-related compounds from this dataset, including cabazitaxel, docetaxel, and paclitaxel, integrating it with the Cancer Dependency Map (https://depmap.org). Interestingly, we found that several cancer types (e.g.: endometrial, breast, and upper aerodigestive tract cancers) with high levels of GMCL1 mRNA exhibited resistance to paclitaxel, cabazitaxel, and/or docetaxel (Figure 3A-C). In contrast, lung cancer cell lines with high GMCL1 mRNA expression did not show such tendencies (Figure 3D). Given that nearly half of lung cancers harbor p53 mutations³⁰, we hypothesized that p53 status may influence GMCL1-mediated Taxol resistance. To further investigate the functional relationship between GMCL1, 53BP1 and p53 in Taxol resistance, we stratified cancer cells based on their p53 status (wild-type vs mutant) and analyzed GMCL1 mRNA expression and 53BP1 protein levels. In cancer cells with wild type p53, high GMCL1 mRNA expression and low 53BP1 protein levels correlated with significant increased resistance to Cabazitaxel and Paclitaxel when compared to cells with low GMCL1 mRNA and high 53BP1 protein levels (Figure 3E). However, this effect was abolished in p53-mutant cells, where GMCL1 status no correlated with Taxol resistance (Figure 3F).

Figure 3.

To further verify the impact of GMCL1 levels on Taxol sensitivity, we performed cell viability and apoptosis assays using cells with wild type or mutant p53. In p53 wild-type cells (MCF7 and U-2OS), Taxol treatment led to a significant reduction in cell viability and an increase in apoptosis in GMCL1-depleted cells compared to cells transfected with non-targeting control siRNA (Figure 4A-D). However, GMCL1 knockdown did not affect cell viability or apoptosis in Taxol-treated cells with mutant p53 or inactivated p53 (HeLa and HEC-1-A, respectively) (Figure 4E-H).

Figure 4.

These observations are consistent with the results shown in Figure 3 and support the hypothesis that Taxol resistance is mediated by GMCL1 through the USP28-p53-53BP1 complex. Specifically, high GMCL1 expression promotes the degradation of 53BP1, maintaining low p53 levels, thereby reducing Taxol-induced cell death in cell that have functional p53.

Discussion

We identify GMCL1, a previously uncharacterized human CRL3 substrate receptor, as a regulator of taxane resistance. Specifically, we found that GMCL1 interacts with and mediates the degradation of 53BP1 during prolonged arrest in M phase. While 53BP1 is well known for its role in double-strand break (DSB) repair via non-homologous end joining (NHEJ)³¹, it also participates to a so-called mitotic stopwatch composed of the 53BP1-USP28-p53 complex that stabilizes p53 during prolonged mitotic arrest^7–9. We showed that GMCL1 controls the levels of 53BP1 and, consequently, those of p53 in mitotic cells, thereby influencing p53 transmission to daughter cells (see model in Figure 4I). This data suggests that GMCL1 functions as a mitotic stress regulator, with potential oncogenic properties in certain contexts.

Cancer database analysis reveals that GMCL1 is overexpressed in multiple cancers, including bladder, brain, breast, kidney, liver, lung, ovary, prostate, skin, and stomach cancers (Supplementary Figure 3). This underscores GMCL1’s role in mitotic regulation and genomic stability, which may vary based on tumor type, genetic background, and additional oncogenic mutations. Recent studies indicate that the MDM2-p53 axis¹⁰, along with the USP28-53BP1-p53 complex, functions as a mitotic timer enforcing G1 arrest following prolonged cell division. Our study identifies GMCL1 as an E3 ligase regulating this pathway by mediating 53BP1 degradation in mitosis. As often is the case for crucial regulatory proteins, 53BP1 is subject to regulation by multiple factors under different cellular circumstances^32–34.

GMCL1 has been primarily studied in the germ cells of D. melanogaster, where we have shown that it forms an active CRL3 complex during mitosis. Our new findings establish a critical role for GMCL1 in somatic cell fate decisions by regulating 53BP1 stability during prolonged mitosis. We found that GMCL1 loss enhances Taxol-induced cell death in p53 wild-type cancers but has no effect in p53-mutant cells (Figure 4I). This suggests that GMCL1 inhibition could be selectively effective in cancers that retain functional p53. These findings position GMCL1 as a key modulator of mitotic stress and Taxol resistance, highlighting its potential as a therapeutic target in cancer treatment.

Materials and methods

Cell culture

Cell lines were purchased from ATCC and were routinely checked for mycoplasma contamination with the MycoStrip Mycoplasma Detection Kit (Invivogen). HEK293T (ATCC CRL-3216), HeLa (ATCC CCL-2) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco). U-2 OS (ATCC HTB-96), HCT-116 (ATCC CCL-247) and HEC-1-A (ATCC HTB-112.NM) cells were maintained in McCoy’s 5A medium (Gibco). MCF7 (ATCC HTB-22) were maintained in Eagle’s Minimum Essential Medium (EMEM) (ATCC). All media were supplemented with 10% fetal bovine serum (FBS) (Corning Life Sciences) and 1% penicillin/streptomycin/L-glutamine (Corning Life Sciences); however, MCF7 was further supplemented with 0.28% human recombinant insulin, zinc solution (11.2 μg/ml, Gibco). All cell lines were maintained at 37 °C and 5% CO2 in a humidified atmosphere.

Plasmids, siRNA, and transfection

Homo sapiens cDNAs were amplified by PCR using KAPA HiFi DNA Polymerase (Kapa Biosystems) and sub-cloned into a variety of vector backbones, including modified pCDNA3.1 vectors containing C-terminal Flag tags, and pLVX-PURO lentiviral vectors containing C-terminal Flag. Site-directed mutagenesis was performed using KAPA HiFi DNA Polymerase (Kapa Biosystems). All cell lines were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific) based on the manufacturer’s recommendation. siRNA oligo transfections were performed using RNAiMax (ThermoFisher Scientific) according to the manufacturer’s instructions.

Virus-mediated gene transfer

For the generation of lentivirus, HEK293T cells were transfected with pLVX constructs carrying the genes of interest, alongside the packaging plasmids pCMV-delta-R8.2 and pCMV-VSV-G. Viral supernatant was harvested 48 hours post-transfection, passed through a 0.45-μm sterile Millex-HV filter unit (Millipore Sigma), and supplemented with polybrene at a final concentration of 8 μg/ml (Sigma). Target cells were infected by replacing their culture medium with the virus-containing supernatant for an 8-hour incubation period. Selection of successfully transduced cells was performed using puromycin at a concentration of 1–2 μg/ml (Sigma).

CRISPR-Cas9 genome editing

CRISPR–Cas9 genome editing techniques were carried out as previously described³⁵ with modifications. In brief, to generate GMCL1-knockout U-2 OS cells, optimal gRNA target sequences closest to the start codon of the genes were designed using the Benchling CRISPR Genome Engineering tool (https://www.benchling.com). For transient Cas9 expression, gRNAs specific for GMCL1 gene was incorporated into the pRP [CRISPR]-Hygro-hCas9-U6 vector, which was obtained from VectorBuilder (https://en.vectorbuilder.com/). The following oligos were used to generate the proper gRNA in the vector: GMCL1 (5’-CGTGCCCCCACGTACCTTCG-3’). To generate GMCL1 2×Flag knock-in HCT116 cells, an optimal gRNA target sequence closest to the genomic target site and a ∼2 kb homologous recombination (HR) donor template was designed using the Benchling CRISPR Genome Engineering tool. The HR donor template was designed to introduce a 2×Flag tag in frame with the C terminus of GMCL1, in the following order: GMCL1-linker-FLAG-linker-FLAG-Stop codon and was purchased from VectorBuilder (https://en.vectorbuilder.com/). The following single gRNA sequence was used for the transient hCas9 expression vector: GMCL1 (5’-AAGTTACAGCAGATATATAA-3’).

Genomic DNA was collected using QuickExtract (Epicentre). Genotyping PCRs were performed with MyTaq HS Red Mix (Bioline), using primers surrounding the genomic target sites. The following primers were used for genotyping: GMCL1 (F: 5’-GCAGGCTTCTGATCTTCCCT-3’, R: 5’-ACTTGTCATCGTCGTCCTTGT-3’), and GMCL1 (F: 5’-GGGTGGGAGTTTGGAGAGTG-3’, R:5’-TCTGGATTTTCTGGGTGACGA-3’). The resulting PCR products were then purified and sequenced to determine the presence of insertion or deletion events. Clones positive to insertion or deletion events were then validated by western blot.

Antibodies

The following antibodies were used: β-actin (1:5,000, Sigma-Aldrich A5441), CUL3 (1:1,000, Bethyl Laboratories A301-109A), FLAG (1:2,000, Sigma-Aldrich F7425), GMCL1 (1:1,000, Proteintech 15575-1-AP), HA (1:2,000, Bethyl Laboratories A190-108A), Histone H3 (1:1,0000, Abcam, ab1791), p21 (1:1,000, Cell Signaling Technology 2947S), p53 (1:1,000, Proteintech 10442-1-AP), pHistone H3 (D2C8) (Ser10, 1:1,000, Cell Signaling Technology, #3377), USP28 (1:1,000, Proteintech 17707-1-AP), 53BP1 (1:2,000, Abcam ab36823), α-tubulin (1:5,000, Sigma-Aldrich T6074).

Drug treatment procedures

Where indicated, cells were treated with 0.4 mg/ml Nocodazole (Sigma-Aldrich M1404) for 16 hours, 10 μM MG132 for 3 hours, 2.5 μM MLN4924 for 3 hours, 100 μg/ml cycloheximide (CHX) for the indicated time.

qRT-PCR

Total RNA was purified using RNeasy mini kits (Qiagen). cDNA was generated using Double Primed EcoDry kits (Takara). The qPCR reaction was carried out using PowerUp SYBR Green (Applied Biosystems) and the Applied Biosystems QuantStudio 3 Real-Time PCR system in a 96-well format. ROX was used as a reference dye for fluorescent signal normalization and for well-to-well optical variations correction. Bar graphs represent the relative ratios of target genes to β-actin housekeeping gene values. For each biological sample, triplicate reactions were analyzed using absolute relative quantification method alongside in-experiment standard curves for each primer set to control for primer efficiency. The oligos used for qRT-PCR analysis were: β-actin (F: 5’-CATGTACGTTGCTATCCAGGC-3’, R: 5’-CTCCTTAATGTCACGCACGAT-3’), GMCL1 (F: 5’-GGAGATTCCTGACCAGAACATTG-3’, R: 5’-CGACTGGGCTTTATCAAGACAT-3), GMCL2 (F: 5’-CCACGCAGCGGGTCTGT, R: 5’-TGGATTTTCTGGGTGACGATTATTT), p21 (F: 5’-TGTCCGTCAGAACCCATGC-3’, R: 5’-AAAGTCGAAGTTCCATCGCTC-3’), NOXA (F: 5’-CCAAGCCGTGACCAAGGAC-3’, R: 5’-CGCCACATTGTGTAGCACCT-3’), PUMA delta (F: 5’-GCCAGATTTGTGAGACAAGAGG-3’, R: 5’-CAGGCACCTAATTGGGCTC-3’).

Immunoprecipitation, immunoblotting, and fractionation

For whole-cell lysates, cells were directly lysed in a buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.2% NP-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 2 mM MgCl₂, 1 mM dithiothreitol (DTT), and 5 mM N-ethylmaleimide (Sigma-Aldrich). When cellular fractionation was performed, it followed a previously established method³⁶. Briefly, cells were lysed in CSK buffer (10 mM PIPES, pH 7.0, 150 mM NaCl, 300 mM sucrose, 0.1% Triton X-100, 3 mM MgCl₂, 1 mM EGTA, 1 mM DTT, and 5 mM N-ethylmaleimide) for 5 minutes. The soluble fraction was collected by centrifugation at 3,000 × g for 3 minutes at 4°C. Cell pellets were subsequently washed in CSK buffer and then lysed in chromatin extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% NP-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 4 mM MgCl₂, 0.5 mM DTT, 5 mM N-ethylmaleimide, and 5 U/mL TurboNuclease (Accelagen)) for 30 minutes. Insoluble debris was removed by centrifugation at 20,000 × g for 15 minutes at 4°C. All buffers were supplemented with protease inhibitors (Complete ULTRA, Roche) and phosphatase inhibitors (PhosSTOP, Roche).

For immunoprecipitation and affinity purification, samples were incubated with FLAG-M2 magnetic beads (Sigma-Aldrich) at 4°C for 2 hours. Beads were thoroughly washed with lysis buffer, and protein elution was performed using either FLAG peptide (Sigma-Aldrich) for mass spectrometry analysis or 1x Laemmli sample buffer for Western Blot analysis.

Immunoblotting

Western blotting was carried out as described previously³⁵. Protein samples were resolved under denaturing and reducing conditions on 4%–12% Bis-Tris gels (NuPAGE) and transferred onto PVDF membranes (Immobilon-P, Millipore). Membranes were blocked with 5% nonfat dried milk, incubated overnight at 4°C with primary antibodies, followed by washes and incubation with HRP-conjugated secondary antibodies (Amersham GE).

Immunoreactive bands were visualized using enhanced chemiluminescence reagents (Pierce) and detected with a ChemiDoc MP imaging system (Bio-Rad). Each Western blot experiment was conducted at least three times to ensure reproducibility, with representative blots shown in the figures.

Cell cycle analysis by flow cytometry

BrdU incorporation and propidium iodide staining were performed following established protocols³⁷. In brief, cells were pulse-labeled with 10 μM BrdU (Merck) for 45 minutes, followed by trypsinization and overnight fixation in ice-cold 70% ethanol. After washing with PBS and subsequently with PBST-BSA (1% BSA, 0.5% Tween-20 in PBS), DNA denaturation was carried out for 30 minutes using 2 N HCl with 0.5% Triton X-100. The cells were then washed in PBS and neutralized by resuspending them in 0.1 M sodium tetraborate (pH 8.5) for 2 minutes. To detect BrdU incorporation, cells were incubated for 1 hour at room temperature with FITC-conjugated anti-BrdU mouse antibodies (BD Transduction Laboratories, 556028) in PBST-BSA. After multiple washes, cells were resuspended in PBST-BSA containing 5 μg/mL propidium iodide (Invitrogen) and 20 μg/mL RNase A (Invitrogen). Flow cytometry analysis of cell cycle distribution was conducted using a CytoFlex Analyzer (Beckman Coulter) and data were processed with FlowJo v10 software (Becton Dickinson).

Mass spectrometry analysis of GMCL1 immunoprecipitations

The eluted anti FLAG-tag antibody purified protein complexes were reduced with 2 μl of 0.2 M DTT for 1 h at 57 °C and subsequently alkylated with 2 μl of 0.5 M iodoacetamide (Sigma) for 45 min at room temperature in the dark. 250 ng of SP3 beads (Cytiva) were added proteins precipitated onto the beads by adding ethanol. Samples were placed in a thermomixer at 25°C for 10 min. Beads were washed three times with 80% ethanol and then digested overnight with 400ng of sequencing grade modified Trypsin (Promega) in 100 mM ammonium bicarbonate. Next, the samples were spun down 21,000 x g for 1 minute. The supernatant was transferred to a new tube while the beads were washed twice with 0.5% acetic acid. The washes were then combined with the supernatant collection.

Samples were acidified with 10% TFA to pH1 and loaded onto a 0.1% TFA equilibrated Pierce C18 spin column using a microcentrifuge. The samples were rinsed twice with 0.1% TFA and twice more using 0.5% acetic acid. Peptides were eluted with 80% acetonitrile in 0.5% acetic acid. The organic solvent was removed using a SpeedVac concentrator and the sample reconstituted in 0.5% acetic acid.

An equal aliquot of each sample was loaded onto a trap column (Acclaim PepMap 100 pre-column, 75 μm × 2 cm, C18, 3 μm, 100 A, Thermo Scientific) connected to an analytical column (EASY-Spray column, 50 m × 75 μm internal diameter, PepMap RSLC C18, 2 μm, 100 A, Thermo Scientific) using the autosampler of an Easy nLC 1200 (Thermo Fisher Scientific) with solvent A consisting of 2% acetonitrile in 0.5% acetic acid and solvent B consisting of 80% acetonitrile in 0.5% acetic acid. The peptide mixture was gradient eluted using the following gradient: 5% solvent B for 5 minutes, 5–35% solvent B in 60 min, 35–45% solvent B in 10 min, followed by 45–100% solvent B in 10 min. The samples were acquired on the Orbitrap Eclipse using the following parameters: full MS spectra resolution of 120,000, an AGC target of 4e5, maximum ion time of 50 ms, scan range from 400 to 1,500 m/z. The MS/MS spectra were collected with the following parameters: a resolution of 30,000, an AGC target of 2e5, maximum ion time of 30 ms, one microscan, 2 m/z isolation window, normalized collision energy (NCE) of 27 and a dynamic exclusion of 30 s. To identify binding partners, all acquired MS2 spectra were searched against a UniProt human database using Sequest HT within Proteome Discoverer 1.4 (Thermo Fisher Scientific).

Fixed modifications were set on cysteine (carbamidomethyl), variable modifications of oxidation on methionine, and deamidation on glutamine and asparagine. The resulting peptide spectra matches and proteins are filtered to better than 1% false discovery rate (FDR) and only proteins with at least two different peptides are reported. Proteins differentially expressed between GMCL1 WT and EK, as determined by SAINT scores with a 5% FDR, were considered significantly enriched interactions when comparing to GMCL1 BTB.

Taxol resistance analysis

Taxol resistance was analyzed across cancer cell lines catalogued in DepMap 24Q2. We integrated TP53 mutation status, GMCL1 RNA and TP53BP1 protein expression information across cells with the PRISM dataset²⁹, to evaluate taxol resistance in the context of GMCL1 or TP53BP1 levels and TP53 mutation.

Cell viability and apoptosis assays

Cells (2500/well) were plated in a 96-well plate. The medium was replaced with 50 µL of medium containing the target siRNA (0.2 µL/well). After 24 hours, an additional 50 µL of the designated reagent was added. Luminescence was measured using BioTek Synergy Neo2 (Agilent) 48 hours post-treatment with CellTiter-Glo 2.0 Cell Viability Assay (Promega) or RealTime-Glo Annexin V Apoptosis and Necrosis Assay (Promega) according to the manufacturer’s protocol.

Quantification and statistical analysis

Data analysis was performed using GraphPad Prism version 10.2.1. For comparisons involving three or more groups, One-way ANOVA followed by Bonferroni’s post hoc test or the Brown-Forsythe and Welch ANOVA test followed by Dunnett’S T3 multiple comparisons test was applied. For bioinformatic data analysis, two-group comparisons were performed using the pairwise Wilcoxon rank-sum test.

Data and code availability

• Original western blot images have been deposited at Mendeley at DOI:10.17632/gj3×6r263d.1 and are publicly available as of the date of publication. https://data.mendeley.com/preview/gj3x6r263d?a=1e452dfc-bf85-472c-83ce-0e997ba6fa40

• The mass spectrometric raw files are accessible at https://massive.ucsd.edu under accession MassIVE MSV000097235 and at www.proteomexchange.org under accession PXD061458. Username: MSV000097235_reviewer, Password: TP53bdg-protein1

Supplemental figures

Supplemental Figure 1.

Supplemental Figure 2.

Supplemental Figure 3.

Acknowledgements

We would like to thank the NYU Proteomics Lab (supported in part by NYU School of Medicine and the Laura and Isaac Perlmutter Cancer Center Support grant P30CA016087 from the National Cancer Institute). We also thank the members of the Pagano Lab for helpful discussions. MP is an investigator with the Howard Hughes Medical Institute and his laboratory is supported by grant R35-GM136250 from the NIH. Y.K. is a recipient of the Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowships and the Uehara Memorial Foundation Postdoctoral fellowship. T.G.R. is grateful for funding from NIH Institutional training grant in Cell Biology (T32GM136542) and HHMI Gilliam Fellowship (GT15758). A. M is supported by US Department of Defense (DoD) (HT9425-24-1-0019). S.K. is a recipient of the Life Sciences Research Foundation (LSRF) Postdoctoral Fellowship and has been an EMBO Long Term Postdoctoral Fellow. G.R. is supported by the Momentum Grant of the Hungarian Academy of Sciences (LP2023-15/2023), EMBO Installation Grant (IG5670-2024) and the HUN-REN Welcome Home and Foreign Researcher Recruitment Grant (KSZF-143/2023).

Additional information

Author contributions

Y.K., G.R. and M.P. conceived, supervised, and coordinated the study. T.G.R. performed all analyses from PRISM and DepMap data. Y.K. performed the mass spectrometric sample preparation, generated CRISIPR KO and KI cell lines, designed plasmid constructs, and performed the cell-based immunoprecipitations, fractionation and protein stability assays. A.M. and J.P. performed the initial mass spectrometric sample preparation and immunoprecipitations. B.M.U and J.O.P performed mass spectrometry analysis. S.F.G. performed structural predictions using AlphaFold3. The manuscript was written with inputs from all authors.

Additional files

Supplementary Table 1