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Contribution of p53-dependent and -independent mechanisms to upregulation of p21 in Fanconi anemia [1]

['Xavier Renaudin', 'Cnrs', 'Université Paris-Saclay', 'Gustave Roussy Institute Cancer Campus', 'Villejuif', 'Equipe Labellisée Ligue Nationale Contre Le Cancer', 'Baraah Al Ahmad Nachar', 'Benedetta Mancini', 'Anna Gueiderikh', 'Noémie Louis-Joseph']

Date: 2024-12

Abnormal expression of the cell cycle inhibitor and p53 target CDKN1A/p21 has been associated with paradoxical outcomes, such as hyperproliferation in p53-deficient cancer cells or hypoproliferation that affects hematopoietic stem cell behavior, leading to bone marrow failure (BMF). Notably, p21 is known to be overexpressed in Fanconi anemia (FA), which is a rare syndrome that predisposes patients to BMF and cancer. However, why p21 is overexpressed in FA and how it contributes to the FA phenotype(s) are still poorly understood. Here, we revealed that while the upregulation of p21 is largely dependent on p53, it also depends on the transcription factor microphthalmia (MITF) as well as on its interaction with the nucleolar protein NPM1. Upregulation of p21 expression in FA cells leads to p21 accumulation in the chromatin fraction, p21 immunoprecipitation with PCNA, S-phase lengthening and genetic instability. p21 depletion in FA cells rescues the S-phase abnormalities and reduces their genetic instability. In addition, we observed that reactive oxygen species (ROS) accumulation, another key feature of FA cells, is required to trigger an increase in PCNA/chromatin-associated p21 and to impact replication progression. Therefore, we propose a mechanism by which p21 and ROS cooperate to induce replication abnormalities that fuel genetic instability.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and a predisposition to cancer caused by biallelic mutations in genes encoding proteins involved in the FANC/BRCA DNA repair pathway. Bone marrow failure has been attributed to the abnormal overexpression of the p53/p21 axis in response to the inherent DNA damage accompanying the loss of the FANC/BRCA pathway, leading to cell cycle arrest and subsequent hematopoietic stem cell exhaustion. Here, we revealed an alternative pathway that contributes to p21 overexpression independent of p53. Indeed, we found that in TP53-null cells, the loss of the FANC pathway still leads to p21 overexpression. We demonstrated that this overexpression is dependent on the transcription factor MITF, which increases CDKN1A mRNA (which encodes p21), and on the NPM1 protein, which directly stabilizes p21. Both mechanisms are independent of DNA damage and/or DNA damage signaling, and therefore, this study provides a shift in the paradigm explaining the bone marrow failure observed in Fanconi anemia. Notably, we demonstrated that, in association with the known higher level of ROS in FA cells, the overexpression of p21 contributes to generating replication stress, which contributes to their genetic instability.

Funding: F.R is supported by grants from La Ligue Contre Le Cancer ( https://www.ligue-cancer.net/ ), Electricité de France ( https://www.edf.fr/ ), Agence nationale pour la Recherche (FANC-Diff) ( https://anr.fr/ ) and SIRIC EpiCure (INCa-DGOS-Inserm-ITMO Cancer_18002) ( https://www.e-cancer.fr/Professionnels-de-la-recherche/Recherche-translationnelle/Les-SIRIC/Le-SIRIC-EpiCURE ). X.R was supported by the Fondation Pour La Recherche Medicale (ARF201909009202) ( https://www.frm.org/fr ) and A.G. was a fellow of the INSERM School Liliane Bettencourt and was supported by a "Course of Excellence in Oncology – Fondation Philanthropia” award ( https://www.inserm.fr/nous-connaitre/ecole-de-linserm-liliane-bettencourt ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: This submission contains all raw data required to replicate the results of our study. Beyond the representative images (Western blot, Immunofluorescence, flow cytometry) present in the main text, the numerical values underlying graphs from all figures can be found in the S1 Table .

Copyright: © 2024 Renaudin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Our findings revealed that in FA cells several pathways/proteins contribute to p21 overexpression beyond the key role of the upregulated p53. Indeed, p21 overexpression in FANC pathway-deficient cell is the result of the convergence of transcription-dependent and transcription-independent events associated with a) DNA damage (ATM-p53 axis), b) nucleolar abnormalities (NPM1), and c) unregulated activation of cellular stress signaling pathways that, in turn, activate the transcription factor microphthalmia (MITF) [ 18 ]. Surprisingly, we observed that p21 overexpression and intracellular ROS in FA cells collectively led to changes in replication characterized by reduced single-cell EdU incorporation. This replicative stress contributes to the lengthening of S phase, hypoproliferative status, and genetic instability, which are major determinants of BMF in this syndrome.

A key biochemical event within the FANC/BRCA pathway is the monoubiquitination of FANCD2 and FANCI mediated by the FANC core complex [ 7 , 8 ]. This occurs during DNA replication in response to delayed or stalled replication forks, facilitating their rescue. Apart from DDR abnormalities, cells lacking functional FANC/BRCA pathways exhibit a proinflammatory phenotype characterized by high intracellular levels of reactive oxygen species (ROS) and constitutive activation of stress signaling pathways [ 9 – 18 ]. These cells exhibit imbalances in ribosome biogenesis associated with altered nucleolar homeostasis and a reduced translation rate [ 19 , 20 ] but also overactivation of the p53-p21 axis, a key determinant of cell cycle progression and proliferation [ 21 – 23 ]. p53-p21 overactivation has been demonstrated to affect hematopoietic stem and progenitor cells (HSPCs) in FA patients and mice, contributing to BMF development [ 22 ]. However, the origins and contributions of these abnormalities to the FA phenotype(s) remain poorly understood.

The primary function of the FANConi/BReast CAncer (FANC/BRCA) tumor suppressor pathway is to ensure accurate chromosome duplication and segregation, a vital process in preventing bone marrow failure (BMF), cancer susceptibility, and the chromosomal fragility syndrome Fanconi anemia (FA) [ 1 – 5 ]. Integrated within the DNA damage response (DDR) network, this pathway orchestrates a set of highly regulated processes, ensuring faithful DNA repair and replication in coordination with cell cycle progression while maintaining genetic stability. Over 20 proteins associated with the FANC/BRCA pathway have been identified, including regulators such as FANCM, the FANCcore complex (FANCA, FANCG, FAAP20, FANCC, FANCE, FANCF, FANCB, FANCL, FAAP100) and the FANCD2/FANCI heterodimer and effectors involved in processes such as homologous recombination, nucleotide excision repair, and G-quadruplex resolution, such as BRCA1/FANCS, BRCA2/FANCD1, PALB2/FANCN, RAD51/FANCR, XPF/ERCC4/FANCQ and BRIP1/BACH1/FANCJ [ 6 ].

Results

Loss of function of the FANC pathway increases p21 expression in both p53-dependent and p53-independent manners The p21 protein, encoded by the CDKN1A gene, is a major p53 target [24], whose transcriptional activity is, in turn, induced by the DDR apex kinases ATM and ATR in response to genotoxic stress [25,26]. Additionally, p53 can be activated in response to nucleolar/ribosomal stress in an ATM/ATR- and phosphorylation-independent manner [27,28]. Both stress conditions have been described in FA, prompting us to investigate the origins of p53-p21 overexpression in FANC pathway-deficient cells. The p21 overexpression was observed following the siRNA-mediated depletion of FANCA or FANCD2 and was normalized by the ectopic expression of the corresponding WT gene in human lymphoblasts (FANCCcorr, Fig 1A; FANCAcorr, S1B Fig). This finding emphasizes that upregulation of the expression of p21 is a direct consequence of the loss of function of an FA-associated protein and is not secondary to additional genetic alterations due to the absence of a functional DDR pathway. PPT PowerPoint slide

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TIFF original image Download: Fig 1. CDKN1A/p21 overexpression in FANCA-deficient cells is a p53-dependent and p53-independent event. (A-F) Representative Western blots showing the levels of p21 and the indicated proteins in (A) WT, FANC-deficient or FANC-corrected lymphoblasts. Ponceau Red staining of the membrane served as a loading control. (B) HCT116 WT or HCT116 TP53-/- cells were transfected with siRNA control (siCtrl) or siRNA targeting FANCA (siFANCA) or FANCD2 (siFANCD2). Actin was used as a loading control. (C) HeLa cells and (D) MRC5 SV40 and MRC5-hTERT cells transfected with siRNA control (siCtrl) or siRNA targeting FANCA (siFANCA) or FANCD2 (siFANCD2). Tubulin was used as a loading control. (E) K562 parental cells and two FANCA KO clones and (F) HeLa Kyoto parental cells and two FANCA KO clones. In C to F, tubulin was used as a loading control. (G) Histograms showing normalized mRNA levels of p21 (CDKN1A) measured by qPCR in HCT116 WT, HCT116 TP53-/- or HeLa cells transfected with siRNA control (siCtrl) or siRNA targeting FANCA (siFANCA) for 72 h. Each point represents an individual experiment. * p<0.05; ** p<0.01. https://doi.org/10.1371/journal.pgen.1011474.g001 Consistent with previous studies, compared with FANC pathway-proficient cells, patient-derived EBV-immortalized lymphoblasts deficient in the FANC core complex (FANCA, FANCC, or FANCG), FANCD2, or FANCD1/BRCA2 exhibited elevated p21 expression (Fig 1A). Accordingly, siRNA-mediated depletion of FANCA or FANCD2 increased the expression of p53 and p21 in HCT116 cells (Fig 1B, left panel). However, unexpectedly, p21 overexpression was also observed, although at an obviously lower level (Fig 1D), in p53-deficient HCT116, HeLa and MRC5-SV40 cells in which FANCA or FANCD2 expression was transiently downregulated by siRNA transfection as well as in K562 cells in which FANCA was knocked out (KO) via the CRISPR/Cas9 approach (Fig 1B–1E). We also generated a new HeLa Kyoto FANCA-KO cell line that does not monoubiquitinate FANCD2 (S1A Fig) and found it overexpresses p21 (Fig 1F). Moreover, to further demonstrate that in FANCA-deficient cells, p21 overexpression is partially independent of p53, we treated the cells with the pifithrin-α, a known inhibitor of p53. While this inhibitor downregulates p21 in WT cells (HSC72corr), we observed that the p21-overexpression in FANCA-/- lymphoblasts is resistant to pifithrin-α (PFT-α) (S1B Fig) suggesting other mechanisms may be involved to sustain p21 overexpression. Subsequent flow cytometry analysis of cell populations stained with propidium iodide (PI, a DNA marker) and an anti-p21 antibody demonstrated that p21 is overexpressed in all cell cycle phases (S1C Fig) independent of the p53 status of the cells. Moreover, in FANCA-deficient cells, p21 overexpression was associated with increased transcription of its mRNA, as determined by qRT–PCR (Fig 1G), suggesting that transcription factors (TFs) other than p53 mediate CDKN1A expression in FA cells. Thus, our findings indicate that in FA, p21 overexpression is predominantly caused by p53, but that in its absence, other pathways can still support p21 upregulation in response to FA deficiency.

MITF participates in the transcriptional induction of p21 in FANCA-deficient cells Having observed (Fig 1G) that CDKN1A expression is increased in p53-deficient cells, we investigated which TFs other than p53 might cooperate to increase its expression. This led us to examine the potential role of the TF microphthalmia (MITF), which is known to induce CDKN1A expression through its binding to the E-box motif CATGTG in its promoter [30]. Indeed, previous studies have shown that MITF is overexpressed in FANC pathway-deficient cells and that it is involved in BMF in FANCA-deficient mice in an as yet undefined manner [18,31,32]. Here, we demonstrated that FANCA depletion in HeLa cells was associated with the induction of MITF, CDKN1A and p21 (Fig 3A to 3C). MITF downregulation in FANCA-depleted HeLa cells strongly reduced CDKN1A expression and p21 protein levels (Fig 3A–3C). In addition, after treating FANCA-deficient p53-proficient lymphoblasts with the MITF inhibitor ML329 [33], we again observed a strong reduction in the intracellular level of p21 (Fig 3D). Thus, our data demonstrate that in FANCA-deficient cells, MITF contributes to the abnormal increase in CDKN1A/p21 expression. Moreover, DNA damage and replication stress did not lead to MITF induction (Fig 3E), confirming that the level of endogenous DNA damage is not sufficient to trigger this axis of response. PPT PowerPoint slide

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TIFF original image Download: Fig 3. MITF is a TF that controls the level of CDKN1A/p21 in cells deficient in p53 and FANCA. (A and B). Histograms showing normalized mRNA levels for MITF (A) and CDKN1A (B) measured by qPCR in HeLa cells transfected with siRNA control (siCtrl), siRNA targeting FANCA (siFANCA) or siRNA targeting FANCA and MITF (siFANCA siMITF) for 72 h. Each point represents an individual experiment. * p<0.05; ** p<0.01. n = 3. (C) Western blot showing the level of p21 expression in HeLa cells 72 hours after siRNA-mediated depletion of FANCA or FANCA and MITF. Tubulin served as a loading control. (D) Western blot showing the level of p21 expression in WT (HSC93) or FANCA-deficient (HSC72) lymphoblasts 18 hours after exposure to the MITF inhibitor ML329. Tubulin served as a loading control. (E) Histograms showing normalized mRNA levels of MITF measured by qPCR in HeLa cells after treatment with HU (1 mM) or MMC (200 ng/mL) for 18 h. https://doi.org/10.1371/journal.pgen.1011474.g003 However, even in p53-deficient and MITF-depleted/inhibited FANCA-deficient cells, p21 protein expression remained above the basal level observed in FANCA-proficient cells (Fig 3C and 3D). Thus, our observations suggest that additional events in cooperation with p53 and MITF increase p21 expression downstream of FANCA deficiency.

Nucleolar/ribosomal stress cooperates with p21 overexpression in FANC pathway-deficient cells We recently demonstrated that FANCA-depleted cells exhibit nucleolar stress, altered ribosome biogenesis and a reduced translational rate [19], and similar findings were reported after FANCI depletion [20]. These abnormalities can lead to increased p21 expression through ATM-independent, p53-dependent and p53-independent mechanisms [34]. Consistently, translation inhibition by puromycin exposure (Fig 4A) or NPM1 downregulation (Fig 4B and 4C), known causes of nucleolar and/or ribosomal stress [35,36], also increased p21 expression in the absence of an active p53 protein. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Nucleolar stress contributes to p21 stabilization in FANCA cells. (A-C) Western blot showing the levels of the indicated proteins in (A) HeLa cells after treatment with 0.1 μg/mL puromycin for 24 h. Tubulin served as a loading control. (B) HCT116 WT and HCT116 TP53-/- cells were transfected with siRNA control (siCtrl), siRNA targeting FANCA (siFANCA), siRNA targeting NPM1 (siNPM1) or siRNA targeting both NPM1 and FANCA (siFANCA/siNPM1). Cells were transfected for 72 h. Beta-actin (Actβ) served as a loading control. (C) HeLa cells were transfected with siRNA control (siCtrl), siRNA targeting FANCA (siFANCA) or siRNA targeting NPM1 and FANCA (siFANCA/siNPM1). Tubulin served as a loading control. (D) Histograms showing normalized mRNA levels for p21 measured by qPCR in HeLa cells transfected with siRNA control (siCtrl) or siRNA targeting FANCA (siFANCA) or FANCA and NPM1 (siFANCA/siNPM1) for 72 h. Each point represents an individual experiment. *p<0.05. n = 3. (E) Immunofluorescence of HeLa cells showing the localization of NPM1. Cells were transfected with either siRNA control (siCtrl) or siRNA targeting FANCA (siFANCA) for 72 h. (F) Histogram showing the mean intensity of NPM1 in the cytosol in at least 100 cells in 3 independent experiments. Statistical differences were assessed by Student’s t test (*p<0.05). The scale bar is 20 μm. (G) PLA analysis between NPM1 and p21 in HeLa cells transfected with siRNA control (siCtrl) or siRNA targeting FANCA (siFANCA) and treated with or without actinomycin D (ACTD) overnight. Representative images of the different conditions in HCT116 WT cells are shown (left), and quantification in HCT116 WT and in HCT116 TP53-/- cells are shown (right). Reactions with single primary antibodies were used as negative controls. (H) Western blot showing the levels of p21 and NPM1 in cells transfected with siRNA control (siCtrl), siRNA targeting FANCA (siFANCA) or FANCA and NPM1 (siFANCA/siNPM1) for 72 h and then treated with cycloheximide (CHX) for the indicated times. Tubulin served as a loading control. The table shows the percentage of p21 protein remaining after 2 h compared to that at time 0. https://doi.org/10.1371/journal.pgen.1011474.g004 In p53-proficient cells, codepletion of NPM1 and FANCA did not exacerbate the overexpression of p21 induced by the downregulation of each protein (Fig 4B, left panel). In contrast, NPM1 depletion strongly reduced the level of p21 associated with FANCA depletion in a p53-deficient background (Fig 4B, right panel, and 4C) without affecting the expression of its encoding RNA (Fig 4D). Nucleolar stress in FA cells was associated with NPM1 displacement in the nucleoplasm ([19], Fig 4E and 4F), where it associated with p21, similar to what was observed in cells treated with the nucleolar stressor actinomycin-D ([34], Fig 4G). Finally, using cycloheximide (CHX), a widely recognized assay used to observe intracellular protein degradation and determine the half-life of a given protein in eukaryotes, we demonstrated that the half-life of p21 was significantly extended by NPM1 in FANCA-deficient cells (Fig 4H). We then tested whether codepletion of both MITF and NPM1 would fully restore p21 in FANCA-depleted cells. Surprisingly, although the p21 level was reduced, it remained at a greater level than that in control cells (S2A Fig). This can be due to the lack of complete depletion of FANCA, NPM1 and/or MITF or to the existence of additional mechanisms contributing to p21 overexpression. This was also observed in an isogenic model of RPE1 cells in which we depleted either NPM1 or MITF in presence or not of p53. Indeed, we showed that depletion of NPM1 was sufficient to reduce the level of p21 independently of p53 status of the cell while MITF was only marginally reducing p21 expression (S2B Fig). Altogether, our observations highlight that p53 and NPM1 cooperate to maintain p21 overexpression. In some situations that could be cell types specific, others transcription factors may sustain an overexpression of p21.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011474

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