Figures and data
Identification of 53BP1 as a GMCL1 interactor
A) Schematics for the immunoprecipitation-mass spectrometry (IP-MS) workflow using wild-type GMCL1 (GMCL1 WT) and mutants (GMCL1 EK and GMCL1 BBO). Color coding: Red, GMCL1; orange, putative substrates/interacting partners; blue, CUL3; green, RBX1; purple, E2 ubiquitin-conjugating enzyme.
B) HEK293T cells were transfected with FLAG-GMCL1 WT, FLAG-GMCL1 EK, or FLAG-GMCL1 BBO. After 24 hours, FLAG-tagged proteins were immunoprecipitated and analyzed by MS/MS. Left panel: proteins enriched with GMCL1 WT vs. BBO; right panel: proteins enriched withGMCL1 EK vs. BBO. Significant interactors were identified using SAINT scores > 0.70 and FDR < 5%.
C) HEK293T cells transfected with empty vector (EV), FLAG-GMCL1 WT, FLAG-GMCL1 BBO, FLAG-GMCL1 WKE_AAA (broadly disrupts the binding to CUL3) and FLAG-GMCL1 EK were treated with MLN4924 (3h). 53BP1 and CUL3 were immunoprecipitated with FLAG beads and analyzed by western blot. Asterisk indicates non-specific bands.
D) HEK293T cells were transfected with EV, FLAG-GMCL1 WT, FLAG-GMCL1 EK, or FLAG-GMCL1 RA. FLAG immunoprecipitations were probed for 53BP1 and CUL3.
E) HEK293T cells were transfected with EV, FLAG-53BP1 WT, FLAG-53BP1 ΔMFF and FLAG-53BP1 IEDI_AAAA. After MLN4924 treatment (3h), GMCL1 was immunoprecipitated and immunoblotted.
F) M phase-synchronized GMCL1 FLAG knock-in HCT116 cells were collected. GMCL1 was immunoprecipitated using FLAG-beads and analyzed by immunoblotting.
GMCL1 targets 53BP1 for degradation during M phase
A) Asynchronous or M phase-synchronized WT or GMCL1 knockout (KO) U2OS cells were collected. Whole-cell extracts (WCE) were prepared using RIPA buffer, and other lysates were fractionated into soluble and chromatin-bound fractions for immunoblotting. Arrow indicates GMCL1-specific bands. Asterisk indicates non-specific bands.
B) Stable U2OS cell lines expressing EV, FLAG-GMCL1 WT, FLAG-GMCL1 EK, or FLAG-GMCL1 RA in a GMCL1 KO background were synchronized into M phase and fractionated for chromatin immunoblotting.
C) Mitotic-synchronized FLAG-GMCL1-expressing cells were collected by shake-off and cultured in fresh FBS-containing medium for 7 h. Daughter cells were treated with CHX at the indicated time points and collected within 7 hours, fractionated into soluble and chromatin-bound fractions, and analyzed by immunoblotting.
GMCL1 expression shows positive correlation with Taxol resistance in cancel cell lines
For each cancer type (A-D) and p53 status (E-F), two visualization methods are presented: Upper panels: Density plots show the distribution of drug responses (log2[cell viability]) to the indicated taxane. Each curve represents the frequency of cell lines exhibiting specific response values. Blue curves represent high GMCL1 expression, while orange curves show low GMCL1 expression. Vertical dashed lines indicate median response values for each group. Rightward shifts of blue curves (high GMCL1) indicate greater resistance.
Lower panels: Boxplots of the same data showing median (horizontal line), interquartile range (box), and distribution range (whiskers). Higher values on the y-axis indicate greater resistance to the drug. Statistical significance is indicated (*p < 0.05; ns: not significant) using Wilcoxon rank-sum test.
In panels E-F, cell lines are stratified by both GMCL1 mRNA and 53BP1 protein levels as ’Low_High’ (low GMCL1/high 53BP1, red) or ’High_Low’ (high GMCL1/low 53BP1, green). Note the significant differences in panels E (wild-type p53) but not in panels F (mutant p53), demonstrating that GMCL1-mediated taxane resistance requires functional p53 signaling.
GMCL1 deficiency sensitizes cancers with wild-type p53 to Taxol-induced apoptosis
MCF7 (A), U2OS (C), HeLa (E), HEC-1-A (G) cells were transfected with GMCL1-targeting siRNAs or non-targeting (NT) control for 72 h. Cells were treated with DMSO or Taxol for 48 h, and cell viability was assessed using the CellTiter-Glo Cell Viability Assay. Error bars represent standard deviation. Apoptosis was measured in the same conditions, i.e., MCF7 (B), U2OS (D), HeLa (F), HEC-1-A (H), using the RealTime-Glo Annexin V Apoptosis and Necrosis Assay. Error bars represent standard deviation.
(I) Overview of GMCL1’s function during the M phase. In cancers with high GMCL1 levels, the CRL3GMCL1-mediated degradation of 53BP1 prevents the formation of the mitotic stopwatch complex, leading to p53 degradation and sustained proliferation. Loss of GMCL1 stabilizes mitotic 53BP1 levels and restores paclitaxel sensitivity.
Mapping 53BP1 binding sites on GMCL1
(A) Predicted structure of GMCL1, domain architecture overview, and comparative analysis of the substrate-binding domain across Drosophila, fish, chicken, mouse, and human. Conserved amino acids are indicated by asterisks, with the human R433 residue highlighted in red.
(B) Schematic representation of 53BP1 domains.
(C) HEK293T cells were co-transfected with FLAG-GMCL1 and either EV, HA-53BP1 WT, or deletion mutants: HA-53BP1 ΔMFF, HA-53BP1 ΔN (lacking the N-terminus of MFF), HA-MFF, HA-MFF ΔOD (oligomerization domain), HA-MFF ΔGAR (glycine-arginine-rich motif), HA-MFF Δ1270-1484, or HA-MFF Δ1370-1484. Immunoprecipitation of 53BP1 was performed using HA beads, followed by immunoblotting of co-purified proteins.
(D) HEK293T cells were transfected with EV or FLAG-GMCL1, together with HA-53BP1 WT or mutants: HA-MFF, HA-53BP1 ΔMFF, HA-53BP1 ΔN (lacking the N-terminus of MFF), HA-53BP1 ΔTudor, and HA-53BP1 ΔC (lacking the C-terminus of MFF). 53BP1 was immunoprecipitated with HA-beads, followed by immunoblotting of co-purified proteins. Asterisk indicates non-specific bands.
(E) To narrow down the GMCL1-binding region on 53BP1, sequential 20-amino-acid deletions within the MFF domain were generated. HEK293T cells were co-transfected with EV or FLAG-GMCL1, along with HA-MFF, HA-MFF Δ1410-1430, and site-specific mutants (every three amino acids mutated within 1410–1430 region of 53BP1). Immunoprecipitation of 53BP1 was conducted with HA-beads, followed by immunoblotting.
(F) HEK293T cells were transfected with FLAG-GMCL1 or FLAG-GMCL2. GMCL1 and GMCL2 were immunoprecipitated with FLAG-beads and analyzed by immunoblotting.
Mitotic stress imprints apoptotic memory in daughter cells
(A) RNA was extracted from FLAG-GMCL1-expressing U2OS cells (as in Figure 2B). p21 and NOXA mRNA levels were quantified by qPCR from three independent experiments. Error bars represent standard deviation.
(B) Stable GMCL1 KO U2OS cells were transduced with lentiviruses expressing EV, FLAG-GMCL1 WT, FLAG-GMCL1 EK, or FLAG-GMCL1 RA. Cells were synchronized in M phase, then released into fresh FBS-containing medium for 20 hours following shake-off. Daughter cells were fractionated into soluble and chromatin-bound fractions and analyzed by immunoblotting.
(C) RNA was extracted from same cells as in (B), and NOXA and PUMA mRNA levels were quantified by qPCR from three independent experiments. Error bars represent standard deviation.
(D) Cell cycle distribution was determined using BrdU pulse and PI staining of asynchronous U2OS WT or FLAG-GMCL1-expressing cells. Error bars represent standard deviation.
Tissue-specific comparison of GMCL1 gene expression levels
The panel shows GMCL1 expression levels in normal cells and cancer cells based on GENT221. The cells in the bottom table indicate where, GMCL1 expression levels were significantly higher in cancer cells (red), while the blue cells indicate cancers that were previously reported in the literature to exhibit Taxol resistance13,38.
