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. 2025 Dec 27;16(6):1213–1221. doi: 10.1002/2211-5463.70189

BMI‐1 modulation and trafficking during M phase in diffuse intrinsic pontine glioma

Banlanjo Umaru 1, Deepak Kumar Mishra 1,3, Shiva Senthil Kumar 1, Hao‐Han Pang 1,4, Brendan Devine 1, Komal Khan 1, Rachid Drissi 1,2,✉
PMCID: PMC13238917  PMID: 41455108

Abstract

BMI‐1 (B‐cell‐specific Moloney murine leukemia virus integration site 1) has been implicated in both normal and cancer cell biology. While the canonical function of BMI‐1 involves epigenetic repression, novel extranuclear functions have been recently reported. In the present study, we demonstrate that the phosphorylation of BMI‐1 in diffuse intrinsic pontine glioma (DIPG) cells occurs in M phase and that it triggers simultaneous translocation of the phosphorylated BMI‐1 to the cytoplasm. This translocation is mediated by the RanGTP‐dependent transporter CRM1, also known as exportin. Furthermore, we uncovered a previously unidentified nuclear export signal (NES) in the BMI‐1 protein, suggesting an active transport type of modified BMI‐1 mediated by CRM1. These findings associate BMI‐1 phosphorylation with its trafficking in M phase. Collectively, this study sheds light on the molecular mechanisms underlying BMI‐1 functions in DIPG, thereby potentially paving the way for the development of targeted therapeutic strategies related to M phase progression.

Keywords: BMI‐1, cell cycle, DIPG, NES, phosphorylation

The schematic illustrates BMI‐1 phosphorylation during M phase, which triggers its translocation from the nucleus to the cytoplasm. In cycling cells, BMI‐1 functions within the PRC1 complex to mediate H2A K119 monoubiquitination. Following PTC596‐induced M phase arrest, phosphorylated BMI‐1 dissociates from PRC1 and is exported to the cytoplasm via its NES through the CRM1 pathway.

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Abbreviations

BMI‐1

B‐cell‐specific Moloney murine leukemia virus integration site 1

CRM1

Chromosomal Region Maintenance 1

DDR

DNA damage response

DIPG

diffuse intrinsic pontine glioma

HTH

helix‐turn‐helix

LMB

Leptomycin‐B

NES

nuclear export signal

PRC1

Polycomb repressive complex 1

BMI‐1 is a highly conserved protein initially identified as a proto‐oncogene that cooperates with c‐MYC in the tumorigenesis of murine B‐cell lymphoma [1]. It is a subunit of the Polycomb repressive complex 1 (PRC1) required for the canonical RING1B‐mediated E3‐ubiquitin ligase activity that catalyzes the ubiquitination of histone H2A at lysine 119 (H2A‐K119Ub). BMI‐1‐associated E3 ubiquitin ligase activity represses multiple gene loci, including the INK4A/ARF locus encoding for two tumor suppressors p16INK4A and p14ARF [2]. BMI‐1 has been implicated in a number of biological functions, including development, cell cycle, DNA damage response (DDR), senescence, stem cell self‐renewal, and differentiation [3]. Furthermore, BMI‐1 is highly expressed in multiple malignancies, and it is involved in therapy resistance, tumorigenesis, metastasis, and cancer recurrence [3, 4]. These functions are attributed to three domains of BMI‐1: the N‐terminal RING finger (RF) and the central helix‐turn‐helix (HTH) domains that are responsible for its interaction with DNA, thereby facilitating gene repression, DDR, and senescence prevention [5, 6, 7, 8]. The C‐terminal PEST domain harbors several phosphorylation sites and confers protein stability and increased oncogenic potential in multiple cancers [9, 10, 11, 12]. Additionally, BMI‐1 contains two nuclear localization sequences (NLS1 and NLS2), with only NLS2 shown to be responsible for its nuclear localization [13, 14].

BMI‐1 is tightly regulated both at the transcriptional and the post‐translational level. While transcriptional regulation has been extensively studied, the role of post‐translational modifications in modulating BMI‐1 function is poorly understood [3]. Recent reports have elucidated novel non‐nuclear functions of BMI‐1. For example, BMI‐1 has been shown to localize to the inner mitochondrial membrane supporting the stability of mitochondrial RNA transcripts and thereby regulates mitochondrial function [14, 15]. BMI‐1 has also been shown to regulate the expression of androgen receptor in prostate cancer, through direct binding in a PRC1‐independent manner [16, 17]. In support of these extranuclear functions, BMI‐1 has been previously shown to be associated with chromatin when hypo‐phosphorylated in G1/S and dissociates from the chromatin in M phase when phosphorylated [18]. Recently, we showed cytoplasmic translocation of phosphorylated BMI‐1 in M phase [19]. However, the process of BMI‐1 translocation from the nucleus to the cytoplasm and its role during M phase remain largely unknown. In the current study, we show that the cytoplasmic translocation of BMI‐1 is M phase specific and may be independent of its function within the PRC1 complex. This translocation is an active process mediated by exportin (CRM‐1). Additionally, we have identified a novel nuclear export signal (NES) sequence within the BMI‐1 protein that allows transport to the cytoplasm by CRM1.

Materials and methods

Cell lines

The primary patient‐derived DIPG cells SU‐DIPG‐IV (RRID:CVCL_IT39), CCHMC‐DIPG‐1, and CCHMC‐DIPG‐2 were cultured as previously described [19]. SU‐DIPG‐IV was authenticated by our collaborator M. Monje (Stanford University). CCHMC‐DIPG‐1 and CCHMC‐DIPG‐2 cells were generated and authenticated by the Drissi laboratory by sequencing. Using the Universal Mycoplasma Detection Kit (30‐1012K; ATCC, Manassas, VA, USA), all cell lines used in this study were confirmed to be negative for mycoplasma contamination. HEK293T cells (RRID: CVCL_0063; ATCC) were purchased from ATCC and used for lentiviral vector packaging.

Reagents

Thymidine (CAS# 50‐89‐5; Sigma, St Louise, MO, USA), Colchicine (CAS# 64‐86‐8; Sigma), Leptomycin‐B (CAS# 87081‐35‐4; Sigma), and Nocodazole (CAS# 31430‐18‐9; Sigma) were reconstituted in sterile water. PTC596 was provided by PTC Therapeutics (South Plainfield, NJ, USA) and was reconstituted in DMSO for in vitro studies.

Generation of ΔNES‐BMI‐1 mutants

The NES deletion (ΔNES‐BMI‐1) cDNA was generated through overlap extension PCR. Wild type (WT) and the NES deletion (ΔNES) BMI‐1 cDNA were cloned into pLVX‐AcGFP1‐N1 Vector (#632154; Clontech, San Jose, CA, USA) to create GFP‐tagged expression vectors. These vectors were transfected into HEK293T cells with additional helper plasmids pMDLg/pRRE (#12251), pMD2.G (#12259), and pRSV‐Rev (#12253) from Addgene (Watertown, MA, USA). Supernatant containing viruses were collected 48 h post‐transfection. The viral supernatants were concentrated by precipitation using polyethylene glycol 6000 (PEG6000) adapted from Kutner et al. [20]. Briefly, PEG6000 (final concentration of ~8.5%) and NaCl (final concentration of ~0.3 m) and PBS (pH 7.2–7.4) were added to the 0.45 μm filtered viral supernatant. The mixture was incubated at 4 °C for at least 1.5 h with gentle mixing every 20–30 min. Following incubation, samples were centrifuged at 7000×  g for 10 min at 4 °C, and the resulting viral pellet was resuspended in 50 mm Tris/HCl (pH 7.4). Viral preparations were aliquoted into screw‐cap tubes, flash‐frozen in dry ice, and stored at −80 °C until use. The viral titer was determined by serial dilution. The concentrated virus was directly added to CCHMC‐DIPG‐1 cells. GFP‐positive cells were sorted and expanded. NES deletion was confirmed through Sanger sequencing.

Cell cycle, proliferation, and apoptosis assays

Cell cycle was analyzed as previously described [19]. Data were analyzed using flowjo v.10 (FlowJo, Ashland, OR, USA) software. Cell proliferation was measured using WST‐1 assay (Takara Bio, MK400, San Jose, CA, USA) as per manufacturer's instructions. WST‐1 reagent was added to each well at a final concentration of 1 : 10, incubated for 1.5 h at 37 °C, and absorbance was measured at 450 nm with 650 nm as the reference wavelength.

Western blotting

Immunoblot assays were performed as previously described [19]. Antibodies used were against BMI‐1 (1 : 1000, Cat# 5856, RRID:AB_10838137; Cell signaling, Danvers, MA, USA), H2AK119Ub (1 : 1000, Cat# 8240S, RRID:AB_10891618; Cell signaling), H3 S10‐P (1 : 1000, Cat# 9706, RRID:AB_331748; Cell signaling), Cyclin B1 (1 : 1000, Cat# 4138, RRID:AB_2072132; Cell signaling), Cleaved caspase‐3 (1 : 1000, Cat# 9661, RRID:AB_2341188; Cell signaling), Total Histon H3 (1 : 1000, Cat# 3638S, RRID:AB_1642229; Cell signaling), Total Histon H2A (1 : 1000, Cat# 3636, RRID:AB_2118801; Cell signaling), β‐Actin (1 : 1000, Cat# 3700, RRID:AB_2242334; Cell signaling). Membranes were stained with corresponding secondary antibodies, Goat anti‐mouse IgG (H + L) Secondary Antibody, HRP (1 : 1000, Cat# 31430, RRID:AB_228307; Thermo Fisher Scientific, Waltham, MA, USA) or Goat anti‐Rabbit IgG (H + L) Secondary Antibody, HRP (1 : 1000, Cat# 31460, RRID: AB_228341, Thermo Fisher Scientific). Bands visualized with ECL were captured using Azurec500 imaging system (Azure Biosystems, Dublin, CA, USA) and quantified using Image Studio Lite (LI‐COR). For the λ‐Phosphatase experiments, 15 μg of protein lysates were treated with λ‐Phosphatase (Cat# P0753S; NEB, Ipswich, MA, USA) according to manufacturer's protocol and then immunoblot was performed. Cytoplasmic and nuclear fractions were isolated as described elsewhere [21].

Immunofluorescence

Immunostaining was performed as described previously [19]. Primary antibodies were used against BMI‐1 (1 : 500, Cat# 6964, RRID:AB_10828713; Cell signaling), RING1B (1 : 500, Cat# 5694, RRID:AB_10705604; Cell signaling), H3 S10‐P (1 : 500, Cat# 9706, RRID:AB_331748; Cell signaling), and Cyclin B1 (1 : 500, Cat# 4138, RRID:AB_2072132; Cell signaling) were stained with corresponding secondary antibodies, Alexa Fluor® 488 AffiniPure™ Donkey Anti‐Rabbit IgG (H + L) (1 : 500, Cat# 711‐545‐152, RRID:AB_2313584; Jackson ImmunoResearch, West Grove, PA, USA) or Alexa Fluor® 594 AffiniPure™ Donkey Anti‐Mouse IgG (H + L) (1 : 500, Cat# 715‐585‐150, RRID:AB_2340854, Jackson ImmunoResearch). For nuclear staining, cells were embedded with mounting media with DAPI (H1200; Vector Laboratories, Newark, CA, USA). Images were captured with a 60× oil objective on a Nikon Eclipse Ti confocal microscope. Quantifications were performed using imagej software (NIH Image).

Statistical analysis

Data from at least two independent experiments with individual technical replicates wherever sapplicable were collected. Representative images or blots are shown. Results are shown as mean ± SD. graphpad prism 8.0.1 was used to perform statistical analysis. One‐ or two‐way ANOVA followed by a post hoc Dunnet's or Tukey test, wherever applicable, was used to analyze the data.

Results

Phosphorylated BMI‐1 is translocated to the cytoplasm in M phase

In our previous studies with PTC596, a mitotic blocker, we showed that treatment with PTC596 led to BMI‐1 phosphorylation and this modification correlated with its translocation to the cytoplasm in cells arrested in M phase [19]. To confirm these observations, we synchronized CCHMC‐DIPG‐1 cells in M phase using nocodazole (Fig. 1A) and observed time‐dependent accumulation of phosphorylated BMI‐1 as the cells arrested in M phase (Fig. 1B). Using previously established mitotic blockers, colchicine or PTC596, we observed an accumulation of phosphorylated BMI‐1 in the cytoplasmic compartment in cells arrested in M phase (Fig. 1C,D). Moreover, these observations were further validated in cell synchronization studies using PTC596 (Fig. S1A–C). Using another DIPG cell line (SU‐DIPG‐IV) treated with either PTC596 or colchicine as well as with λ‐phosphatase, we showed that modification of BMI‐1 occurs via phosphorylation (Fig. S1D). Of note, we used Cyclin B1 as a surrogate marker for identifying cells in the G2/M phase in both immunoblot and immunofluorescence (IF) assays. Consistent with previous findings, Cyclin B1 accumulates in the cytoplasm as cells approach prophase [22, 23, 24].

Fig. 1.

Fig. 1

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Phosphorylation and translocation of BMI‐1 occurs in M phase. (A) Scheme for the cell synchronization studies (top) and corresponding cell cycle analysis by flow cytometry (bottom). The percentage of cells in each cell cycle is indicated. (B) Immunoblot analysis of BMI‐1 at the indicated timepoints. The arrow indicates phosphorylated BMI‐1. β‐Actin is used as the loading control. MW indicates molecular weight marker in kDa. (C) Immunoblot analysis of BMI‐1, Cyclin B1 and histone H3 S10‐P in the cytoplasmic (C) and nuclear (N) fractions when treated with PTC596 (100 nm for 24 h) or colchicine (100 ng·mL−1 for 24 h). β‐Actin and total H3 served as loading controls. Arrow indicates phosphorylated BMI‐1. MW indicates Molecular Weight marker in kDa. (D) Representative immunofluorescence images showing DAPI (blue), Cyclin B1 (green) and BMI‐1 (red) in cells treated with PTC596 or colchicine at the indicated doses for 24 h. White arrows indicate the cells with cytoplasmic localization of BMI‐1. The scale bar is 100 μm. (E) immunoblot analysis of RING1B in the cytoplasmic (C) and nuclear (N) fractions in cells treated with PTC596 (100 nm for 24 h) or colchicine (100 ng·mL−1 for 24 h). MW indicates molecular weight marker in kDa. (F) Representative immunofluorescence images showing DAPI (blue), H3‐S10‐P (green) and RING1B (red) in cells treated with 100 nm PTC596 for 24 h. White arrows indicate the cells with cytoplasmic localization of BMI‐1. The scale bar is 100 μm. (G) Representative immunofluorescence images showing DAPI (blue), H3‐S10‐P (green) and RING1B (red) in cells treated with 100 nm PTC596 for 24 h. Yellow arrows indicate the cells with RING1B still localized within DNA. All experiments were conducted in CCHMC‐DIPG‐1 cells at n = 2. The data are presented as mean ± SD. P values are indicated [*P < 0.05; ns, not significant (P > 0.05)] using a two‐tailed Student's t‐test.

Given that BMI‐1 is a crucial component of the PRC1 complex, we aimed to investigate whether its phosphorylation and translocation influence the trafficking of the entire complex. Interestingly, RING1B, the catalytic subunit of the PRC1 complex, was not translocated to the cytoplasm when treated with PTC596 or colchicine (Fig. 1E). Furthermore, IF analysis validated these findings by confirming cytoplasmic translocation of BMI‐1 and not of RING1B (Fig. 1F,G) in M phase cells as evidenced by H3 S10‐P positive staining. Together, these data suggest that BMI‐1 phosphorylation and translocation to the cytoplasm in M phase may be independent of its function within the PRC1 complex.

Transport of modified BMI‐1 to the cytoplasm is CRM1 dependent

When treated with PTC596, we observed a small percentage of cells showing both cytoplasmic and nuclear BMI‐1 distribution (Fig. S2A), suggesting that BMI‐1 may be actively transported from the nucleus to the cytoplasm during M phase. Nuclear export of the majority of proteins to the cytoplasm is directed by the nuclear export receptor CRM1 or exportin‐1 [25, 26]. Leptomycin‐B (LMB), a potent selective inhibitor of CRM1‐mediated transport, has been previously reported to inhibit nuclear export of Cyclin B1 in a time‐dependent manner [23, 24]. We observed a gradual decrease in cytoplasmic BMI‐1 and a consequent increase in the nuclear levels in a time‐dependent manner when treated with LMB (Fig. 2A). Interestingly, when treated together with PTC596, LMB inhibited the cytoplasmic translocation of phosphorylated BMI‐1 in M phase (Fig. 2B,D and Fig. S2B). Moreover, LMB treatment interfered with PTC596‐induced M phase arrest (Fig. 2C). Together, these data suggest that the BMI‐1 translocation to the cytoplasm in M phase is an active process and is CRM1‐dependent.

Fig. 2.

Fig. 2

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M phase translocation of BMI‐1 is an active process. (A) Immunoblot analysis of BMI‐1, Cyclin B1 and H3 S10‐P in the cytoplasmic and nuclear fractions of cells treated with leptomycin B (LMB) for the indicated time points. β‐Actin and total H3 served as the loading controls. MW indicates molecular weight marker in kDa. (B) Scheme for the combination experiment of LMB with PTC596 (top) and corresponding immunoblot analysis of BMI‐1 and H3 S10‐P in the cytoplasmic and nuclear fractions (bottom). β‐Actin and total H3 served as loading controls. MW indicates molecular weight marker in kDa. (C) Cell cycle analysis by flow cytometry of cells treated with LMB and PTC596. The percentage of cells in each cell cycle phase is indicated. (D) Representative immunofluorescence images showing DAPI (blue), BMI‐1 (green) and H3 S10‐P (red) in cells treated with PTC596 and LMB based on scheme in (B). White arrows indicate the cells with cytoplasmic localization of BMI‐1. The scale bar is 100 μm. All experiments were conducted in CCHMC‐DIPG‐1 cells at n = 2. The data are presented as mean ± SD. P values are indicated (*P < 0.05) using one‐way ANOVA with Dunnett's post hoc test.

Characterization of functional NES motif in BMI‐1

CRM1‐mediated transport requires a leucine‐rich sequence in the target cargo proteins that have been characterized as nuclear export signals (NES) [27, 28]. Using an NES prediction pipeline [29], we identified a previously unidentified putative NES sequence, 175‐LRKFLRSKMDI‐185 within the HTH domain (Fig. S3A). This sequence conforms to the NES consensus Class 1C sequence (φ‐XXX‐φ‐XXX‐φ‐X‐φ) [30, 31] and is highly conserved across multiple species (Fig. 3A,B). Additionally, mutations in the CRM1 gene have been previously shown to interfere with the transport function of the CRM1 protein [32]. We confirmed that CCHMC‐DIPG‐1 cells do not harbor any mutations in the CRM1 gene. To examine whether the putative NES sequence is functional, we ectopically expressed ΔNES‐BMI‐1 (D1‐ΔNES‐BMI‐1) as well as the BMI‐1 overexpression control (D1‐BMI‐1) in CCHMC‐DIPG‐1 cells (Fig. S3B). When treated with PTC596, we observed both a decrease in phosphorylation in D1‐ΔNES‐BMI‐1 and a defect in phosphorylated BMI‐1 cytoplasmic translocation compared to D1‐BMI‐1. Importantly, the ectopic expression of ΔNES‐BMI‐1 did not affect the phosphorylation and cytoplasmic translocation of endogenous BMI‐1 (Figs 3C and S3A). Moreover, the ectopic expression of ΔNES‐BMI‐1 did not affect the M phase arrest of cells treated with PTC596 (Fig. 3C,D), suggesting that a novel NES sequence identified in BMI‐1 may be specifically involved in conformational change and translocation of phosphorylated BMI‐1 in M phase.

Fig. 3.

Fig. 3

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Identification and characterization of NES in BMI‐1. (A) Output of the NES prediction pipeline NetNES 1.1 showing putative NES signals in the BMI‐1 protein sequence (HMM, hidden Markov model algorithm; NES score = combination of NN and HMM algorithms; NN, neural network algorithm). A signal above the threshold signifies a highly probable NES hit. (B) Alignment of the identified BMI‐1 NES sequence in multiple species, verified NES sequences in TERT and SNUPN and the NES consensus sequences showing high homology. The consensus sequences for each species were obtained using netnes 1.1 prediction software and corresponded to the consensus (φ‐XXX‐φ‐XXX‐φ‐X‐φ, where φ represents the amino acids L, I, V, M, or F, while X represents any amino acid). The obtained sequences were manually aligned. (C) Immunoblot analysis of BMI‐1, H3 S10‐P from cytoplasmic (C) and nuclear (N) fractions of D1‐BMI‐1 and D1‐ΔNES‐BMI‐1 cells treated with PTC596. β‐Actin and total H3 served as loading controls. BMI‐1‐GFP is ectopically expressed in D1‐BMI‐1 and D1‐ΔNES‐BMI‐1. Arrows indicate phosphorylated BMI‐1, n = 3. MW indicates Molecular Weight marker in kDa. (D) Cell cycle analysis by flow cytometry of D1‐WT‐BMI‐1 and D1‐ΔNES‐BMI‐1 cells treated with DMSO or PTC596 (100 nm for 24 h). The percentage of cells in each cell cycle phase is indicated.

Discussion

Diffuse Intrinsic Pontine Glioma (DIPG) represents one of the most aggressive malignancies of the central nervous system, predominantly affecting children with a median overall survival of less than 1 year. Hence, there is an urgent need to develop novel therapies that not only improve outcome but mitigate long‐term complications in children with DIPG. We have previously identified BMI‐1 as a potential therapeutic target in DIPG and have shown that BMI‐1 is highly expressed in DIPG tumors regardless of DIPG subtype [33]. The protein BMI‐1, a critical component of the Polycomb Repressive Complex 1 (PRC1), is a proto‐oncogene implicated in development, stemness of normal and malignant cells, and self‐renewal. Elucidating the molecular mechanisms underlying BMI‐1 functions in DIPG‐M phase progression will contribute to the development of targeted therapeutic strategies.

We have previously reported the phosphorylation of BMI‐1 in M phase and the simultaneous translocation to the cytoplasm of modified BMI‐1 when DIPG cells were treated with PTC596 [19]. Our present study demonstrates that the phosphorylation and translocation of BMI‐1 are independent of the PRC1 complex. Interestingly, the translocation is an active process mediated by CRM1, an essential mediator of nuclear protein export. Voncken et al. [18, 34] using osteosarcoma cells, showed that BMI‐1 was phosphorylated at the G2/M phase of the cell cycle and dissociates from PRC1 complex proteins on chromatin. However, in this study, we observed colocalization of RING1B, the catalytic subunit of the PRC1 complex, with chromatin and no cytoplasmic translocation during M phase in our DIPG cells treated either with colchicine or PTC596 (Fig. 1E,F). This suggests that the phosphorylation and translocation to the cytoplasm of BMI‐1 may be independent of its function within the PRC1 complex and indicate a non‐canonical role of BMI‐1 in DIPG cells. Moreover, we have previously demonstrated high expression levels of BMI‐1 and normal levels of RING1B in DIPG tumor tissues and across all subtypes of patient‐derived DIPG neurospheres [33], suggesting extranuclear PRC1‐independent and non‐canonical functions of BMI‐1. Further studies are required to elucidate these extranuclear functions of phosphorylated BMI‐1 in DIPG. Extranuclear BMI‐1 has been shown to localize to the inner mitochondrial membrane to stabilize the mitochondrial RNA, thereby regulating the electron transport chain, which enhances oxidative phosphorylation and ATP synthesis and prevents aberrant ROS production [14, 15].

During mitosis, the RanGTPases, including CRM1, control multiple cellular processes involving nuclear transport, mitotic checkpoints, spindle assembly, and post‐mitotic nuclear envelope reassembly [25, 26]. Here, we show that phosphorylated BMI‐1 is transported actively from the nucleus to the cytoplasm via CRM1. A comprehensive analysis of the mitosis events is required to determine the timing of BMI‐1 phosphorylation and consequent transport from the nucleus to the cytoplasm during M phase. We have previously postulated that BMI‐1 phosphorylation occurs in early metaphase before induction of the spindle assembly checkpoint (SAC) and the anaphase promoting complex (APC/CCDC20) [19], albeit Kim et al. [35] have suggested that PTC596 directly inhibits the APC/CCDC20 leading to persistent cyclin‐dependent kinase (CDK)1/2 activity and BMI‐1 hyper‐phosphorylation, as well as reduced PRC1 activity. In this study, we identified for the first time an NES sequence in BMI‐1 protein involved in its CRM1‐mediated nuclear export. Indeed, previous studies have reported an NES‐dependent nuclear export of Cyclin B1 by CRM1 [22, 23, 24]. Moreover, treatment with LMB, which was shown to inhibit interactions of CRM1 with NES [36], coupled with Cyclin B1 accumulation in the nucleus led to cell cycle arrest in S and G2 [24]. Furthermore, LMB inhibition of CRM1 was shown to disrupt mitotic progression and chromosome segregation [37]. As expected, we observed an inhibition of PTC596‐induced M phase arrest when cells were treated with LMB, thus interfering with modified BMI‐1 translocation. Future studies with genetic ablation of CRM1 will be required to validate this CRM1‐specific involvement in phosphorylated BMI‐1 translocation.

Several questions remain to be answered to elucidate the exact role of BMI‐1 phosphorylation in tumor survival and progression. We are currently performing further studies to determine the phosphorylation sites, upstream kinases, effects of phosphorylation on structural modification of BMI‐1, new binding partners of modified BMI‐1, and ultimately the extranuclear locations and non‐canonical functions of BMI‐1.

Conclusion

In conclusion, our findings indicate a potential non‐canonical function of BMI‐1 in DIPG during M phase. BMI‐1 undergoes phosphorylation during the M phase, dissociates from the PRC1 complex, and subsequently actively translocates to the cytoplasm. Future studies including site‐directed mutagenesis of Lysine residues in the NES domain should elucidate its role in BMI‐1 transport. Additionally, exploring and targeting the BMI‐1 phosphorylation sites is necessary to uncover the extranuclear locations and functions of phosphorylated BMI‐1 in DIPG.

Conflict of interest

The authors declare no conflict of interests.

Author contributions

DKM, BU, SSK, H‐HP, and RD contributed to conception and design. DKM, SSK, BU, H‐HP, and RD contributed to development of methodology. DKM, SSK, BD, KK, and UB contributed to acquisition of data. DKM, SSK, BU, and RD contributed to analysis and interpretation of data. All authors contributed to writing, review, and/or revision of the manuscript. BD and KK contributed to administrative, technical, or material support. RD contributed to study supervision.

Supporting information

Fig. S1. Further evidence indicating that BMI‐1 undergoes phosphorylation and translocation specifically during the M phase, as referenced in Fig. 1.

Fig. S2. Additional evidence that BMI‐1 translocation during M phase is an active process, related to Fig. 2.

Fig. S3. Depiction of the newly identified NES domain within BMI‐1 and Sanger sequencing analysis of BMI‐1 constructs bearing a truncated NES domain.

FEB4-16-1213-s001.pdf (416.9KB, pdf)

Acknowledgements

This work was supported by the Department of Defense grant 13709017, the National Center for Advancing Translational Sciences grant UM1TR004548, PTC Therapeutics, Inc., and Nationwide Children's Hospital, Columbus, OH. We thank M. Monje (Stanford University) for kindly providing us with SU‐DIPG‐IV cells. We thank PTC Therapeutics, Inc. for providing PTC596.

Data accessibility

Data were analyzed using flowjo v.10, image studio lite, graphpad prism 10, and NIH Image J. This paper does not report original code. Scripts and code utilized in this study are available from the lead contact upon request.

References

  • 1. van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Gulden H and Berns A (1991) Identification of cooperating oncogenes in E mu‐myc transgenic mice by provirus tagging. Cell 65, 737–752. [DOI] [PubMed] [Google Scholar]
  • 2. Jacobs JJ, Scheijen B, Voncken JW, Kieboom K, Berns A and van Lohuizen M (1999) Bmi‐1 collaborates with c‐Myc in tumorigenesis by inhibiting c‐Myc‐induced apoptosis via INK4a/ARF. Genes Dev 13, 2678–2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Bhattacharya R, Mustafi SB, Street M, Dey A and Dwivedi SK (2015) Bmi‐1: at the crossroads of physiological and pathological biology. Genes Dis 2, 225–239. doi: 10.1016/j.gendis.2015.04.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Sahasrabuddhe AA (2016) BMI1: a biomarker of hematologic malignancies. Biomark Cancer 8, 65–75. doi: 10.4137/bic.S33376 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Facchino S, Abdouh M, Chatoo W and Bernier G (2010) BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery. J Neurosci 30, 10096–10111. doi: 10.1523/JNEUROSCI.1634-10.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Ginjala V, Nacerddine K, Kulkarni A, Oza J, Hill SJ, Yao M, Citterio E, van Lohuizen M and Ganesan S (2011) BMI1 is recruited to DNA breaks and contributes to DNA damage‐induced H2A ubiquitination and repair. Mol Cell Biol 31, 1972–1982. doi: 10.1128/MCB.00981-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Ismail IH, Andrin C, McDonald D and Hendzel MJ (2010) BMI1‐mediated histone ubiquitylation promotes DNA double‐strand break repair. J Cell Biol 191, 45–60. doi: 10.1083/jcb.201003034 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Itahana K, Zou Y, Itahana Y, Martinez JL, Beausejour C, Jacobs JJ, Van Lohuizen M, Band V, Campisi J and Dimri GP (2003) Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi‐1. Mol Cell Biol 23, 389–401. doi: 10.1128/mcb.23.1.389-401.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Kim J, Hwangbo J and Wong PKY (2011) p38 MAPK‐mediated Bmi‐1 down‐regulation and defective proliferation in ATM‐deficient neural stem cells can Be restored by Akt activation. PLoS One 6, e16615. doi: 10.1371/journal.pone.0016615 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Liu Y, Liu F, Yu H, Zhao X, Sashida G, Deblasio A, Harr M, She QB, Chen Z, Lin HK et al. (2012) Akt phosphorylates the transcriptional repressor Bmi1 to block its effects on the tumor‐suppressing Ink4a‐Arf locus. Sci Signal 5, ra77. doi: 10.1126/scisignal.2003199 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Nacerddine K, Beaudry JB, Ginjala V, Westerman B, Mattiroli F, Song JY, Van Der Poel H, Ponz OB, Pritchard C, Cornelissen‐Steijger P et al. (2012) Akt‐mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer. J Clin Invest 122, 1920–1932. doi: 10.1172/JCI57477 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Yadav AK, Sahasrabuddhe AA, Dimri M, Bommi PV, Sainger R and Dimri GP (2010) Deletion analysis of BMI1 oncoprotein identifies its negative regulatory domain. Mol Cancer 9, 158. doi: 10.1186/1476-4598-9-158 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Cohen KJ, Hanna JS, Prescott JE and Dang CV (1996) Transformation by the Bmi‐1 oncoprotein correlates with its subnuclear localization but not its transcriptional suppression activity. Mol Cell Biol 16, 5527–5535. doi: 10.1128/MCB.16.10.5527 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Mustafi SB, Aznar N, Dhar Dwivedi SK, Chakraborty PK, Basak R, Mukherjee P, Ghosh P and Bhattacharya R (2016) Mitochondrial BMI1 maintains bioenergetic homeostasis in cells. FASEB J 30, 4042–4055. doi: 10.1096/fj.201600321r [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Liu J, Cao L, Chen J, Song S, Lee IH, Quijano C, Liu H, Keyvanfar K, Chen H, Cao L‐Y et al. (2009) Bmi1 regulates mitochondrial function and the DNA damage response pathway. Nature 459, 387–392. doi: 10.1038/nature08040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Zhu S, Zhao D, Li C, Li Q, Jiang W, Liu Q, Wang R, Fazli L, Li Y, Zhang L et al. (2020) BMI1 is directly regulated by androgen receptor to promote castration‐resistance in prostate cancer. Oncogene 39, 17–29. doi: 10.1038/s41388-019-0966-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Zhu S, Zhao D, Yan L, Jiang W, Kim JS, Gu B, Liu Q, Wang R, Xia B, Zhao JC et al. (2018) BMI1 regulates androgen receptor in prostate cancer independently of the polycomb repressive complex 1. Nat Commun 9, 500. doi: 10.1038/s41467-018-02863-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Voncken JW, Schweizer D, Aagaard L, Sattler L, Jantsch MF and van Lohuizen M (1999) Chromatin‐association of the polycomb group protein BMI1 is cell cycle‐regulated and correlates with its phosphorylation status. J Cell Sci 112, 4627–4639. [DOI] [PubMed] [Google Scholar]
  • 19. Senthil Kumar S, Sengupta S, Zhu X, Mishra DK, Phoenix T, Dyer L, Fuller C, Stevenson CB, DeWire M, Fouladi M et al. (2020) Diffuse intrinsic pontine glioma cells are vulnerable to mitotic abnormalities associated with BMI‐1 modulation. Mol Cancer Res 18, 1711–1723. doi: 10.1158/1541-7786.MCR-20-0099 [DOI] [PubMed] [Google Scholar]
  • 20. Kutner RH, Zhang XY and Reiser J (2009) Production, concentration and titration of pseudotyped HIV‐1‐based lentiviral vectors. Nat Protoc 4, 495–505. doi: 10.1038/nprot.2009.22 [DOI] [PubMed] [Google Scholar]
  • 21. Suzuki K, Bose P, Leong‐Quong RY, Fujita DJ and Riabowol K (2010) REAP: a two minute cell fractionation method. BMC Res Notes 3, 294. doi: 10.1186/1756-0500-3-294 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Clute P and Pines J (1999) Temporal and spatial control of cyclin B1 destruction in metaphase. Nat Cell Biol 1, 82–87. doi: 10.1038/10049 [DOI] [PubMed] [Google Scholar]
  • 23. Hagting A, Jackman M, Simpson K and Pines J (1999) Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation‐dependent nuclear import signal. Curr Biol 9, 680–689. doi: 10.1016/s0960-9822(99)80308-x [DOI] [PubMed] [Google Scholar]
  • 24. Toyoshima F, Moriguchi T, Wada A, Fukuda M and Nishida E (1998) Nuclear export of cyclin B1 and its possible role in the DNA damage‐induced G2 checkpoint. EMBO J 17, 2728–2735. doi: 10.1093/emboj/17.10.2728 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Forbes DJ, Travesa A, Nord MS and Bernis C (2015) Nuclear transport factors: global regulation of mitosis. Curr Opin Cell Biol 35, 78–90. doi: 10.1016/j.ceb.2015.04.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Weis K (2003) Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 112, 441–451. doi: 10.1016/s0092-8674(03)00082-5 [DOI] [PubMed] [Google Scholar]
  • 27. Dong X, Biswas A, Süel KE, Jackson LK, Martinez R, Gu H and Chook YM (2009) Structural basis for leucine‐rich nuclear export signal recognition by CRM1. Nature 458, 1136–1141. doi: 10.1038/nature07975 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Xu D, Farmer A, Collett G, Grishin NV and Chook YM (2012) Sequence and structural analyses of nuclear export signals in the NESdb database. Mol Biol Cell 23, 3677–3693. doi: 10.1091/mbc.e12-01-0046 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. La Cour T, Kiemer L, Mølgaard A, Gupta R, Skriver K and Brunak S (2004) Analysis and prediction of leucine‐rich nuclear export signals. Protein Eng Des Sel 17, 527–536. doi: 10.1093/protein/gzh062 [DOI] [PubMed] [Google Scholar]
  • 30. Fung HYJ, Fu S‐C, Brautigam CA and Chook YM (2015) Structural determinants of nuclear export signal orientation in binding to exportin CRM1. elife 4, e10034. doi: 10.7554/elife.10034 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Fung HYJ, Fu S‐C and Chook YM (2017) Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals. elife 6, e23961. doi: 10.7554/elife.23961 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Baumhardt JM, Walker JS, Lee Y, Shakya B, Brautigam CA, Lapalombella R, Grishin N and Chook YM (2020) Recognition of nuclear export signals by CRM1 carrying the oncogenic E571K mutation. Mol Biol Cell 31, 1879–1891. doi: 10.1091/mbc.e20-04-0233 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Kumar SS, Sengupta S, Lee K, Hura N, Fuller C, DeWire M, Stevenson CB, Fouladi M and Drissi R (2017) BMI‐1 is a potential therapeutic target in diffuse intrinsic pontine glioma. Oncotarget 8, 62962–62975. doi: 10.18632/oncotarget.18002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Voncken JW, Niessen H, Neufeld B, Rennefahrt U, Dahlmans V, Kubben N, Holzer B, Ludwig S and Rapp UR (2005) MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1. J Biol Chem 280, 5178–5187. doi: 10.1074/jbc.M407155200 [DOI] [PubMed] [Google Scholar]
  • 35. Kim MJ, Cao L, Sheedy J, Risher N, Dumble M, Lee C‐S, Sydorenko N, Baiazitov R, Du W, Moon Y‐C et al. (2014) Abstract 5517: PTC596‐induced Bmi1 hyper‐phosphorylation via Cdk1/2 activation resulting in tumor stem cell depletion. Cancer Res 74, 5517. doi: 10.1158/1538-7445.Am2014-5517 [DOI] [Google Scholar]
  • 36. Fornerod M, Ohno M, Yoshida M and Mattaj IW (1997) CRM1 is an export receptor for leucine‐rich nuclear export signals. Cell 90, 1051–1060. doi: 10.1016/s0092-8674(00)80371-2 [DOI] [PubMed] [Google Scholar]
  • 37. Arnaoutov A, Azuma Y, Ribbeck K, Joseph J, Boyarchuk Y, Karpova T, McNally J and Dasso M (2005) Crm1 is a mitotic effector of ran‐GTP in somatic cells. Nat Cell Biol 7, 626–632. doi: 10.1038/ncb1263 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Further evidence indicating that BMI‐1 undergoes phosphorylation and translocation specifically during the M phase, as referenced in Fig. 1.

Fig. S2. Additional evidence that BMI‐1 translocation during M phase is an active process, related to Fig. 2.

Fig. S3. Depiction of the newly identified NES domain within BMI‐1 and Sanger sequencing analysis of BMI‐1 constructs bearing a truncated NES domain.

FEB4-16-1213-s001.pdf (416.9KB, pdf)

Data Availability Statement

Data were analyzed using flowjo v.10, image studio lite, graphpad prism 10, and NIH Image J. This paper does not report original code. Scripts and code utilized in this study are available from the lead contact upon request.

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