Mcl-1 deficiency in murine livers leads to nuclear polyploidisation and mitotic errors: Implications for hepatocellular carcinoma

Background & Aims Mcl-1, an antiapoptotic protein overexpressed in many tumours, including hepatocellular carcinoma (HCC), represents a promising target for cancer treatment. Although Mcl-1 non-apoptotic roles might critically influence the therapeutic potential of Mcl-1 inhibitors, these functions remain poorly understood. We aimed to investigate the effects of hepatic Mcl-1 deficiency (Mcl-1Δhep) on hepatocyte ploidy and cell cycle in murine liver in vivo and the possible implications on HCC. Methods Livers of young Mcl-1Δhep and wild-type (WT) mice were analysed for ploidy profile, mitotic figures, in situ chromosome segregation, gene set enrichment analysis and were subjected to two-thirds partial hepatectomy to assess Mcl-1 deficiency effect on cell cycle progression in vivo. Mcl-1Δhep tumours in older mice were analysed for ploidy profile, chromosomal instability, and mutational signatures via whole exome sequencing. Results In young mice, Mcl-1 deficiency leads to nuclear polyploidy and to high rates of mitotic errors with abnormal spindle figures and chromosome mis-segregation along with a prolonged spindle assembly checkpoint activation signature. Chromosomal instability and altered ploidy profile are observed in Mcl-1Δhep tumours of old mice as well as a characteristic mutational signature of currently unknown aetiology. Conclusions Our study suggests novel non-apoptotic effects of Mcl-1 deficiency on nuclear ploidy, mitotic regulation, and chromosomal segregation in hepatocytes in vivo. In addition, the Mcl-1 deficiency characteristic mutational signature might reflect mitotic issues. These results are of importance to consider when developing anti-Mcl-1 therapies to treat cancer. Impact and implications Although Mcl-1 inhibitors represent promising hepatocellular carcinoma treatment, the still poorly understood non-apoptotic roles of Mcl-1 might compromise their successful clinical application. Our study shows that Mcl-1 deficiency leads to nuclear polyploidy, mitotic errors, and aberrant chromosomal segregation in hepatocytes in vivo, whereas hepatocellular tumours spontaneously induced by Mcl-1 deficiency exhibit chromosomal instability and a mutational signature potentially reflecting mitotic issues. These results have potential implications for the development of anti-Mcl-1 therapies to treat hepatocellular carcinoma, especially as hyperproliferative liver is a clinically relevant situation.


Introduction
Liver cancer is one of the leading causes of cancer-related death worldwide, and hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers. 1,2Myeloid cell leukaemia sequence 1 (Mcl-1), an anti-apoptotic Bcl-2 family member, is overexpressed in many tumour types, including HCC. 3 Mcl-1 overexpression is implicated in cancer drug-resistance, as blocking apoptosis allows cells to survive cytotoxic chemotherapeutic challenge. 3Therefore, Mcl-1 inhibitors targeting its prosurvival function are under development. 4,5Counterintuitively, we previously showed that a mouse model with a hepatocyte specific deletion of Mcl-1 (Mcl-1 Dhep ) spontaneously develops HCC with an incidence of 50% at 12 months of age.7][8] It was proposed that the proapoptotic environment and concomitant proliferation impose a higher DNA replication rate, increasing the risk of replication-associated DNA damage, leading to genetic instability and tumorigenesis. 8This suggests that Mcl-1 deficiency-associated apoptosis has oncogenic potential in the liver but also in the gastrointestinal tract. 91][12][13][14][15][16] The complexity of Mcl-1 regulation and its multiple functions, particularly its poorly understood non-apoptotic roles, represent challenges to the successful clinical application of Mcl-1 inhibitors. 15,17Thus, it is crucial to unravel further the non-apoptotic roles of Mcl-1 in the liver in vivo.
9][20] Polyploid hepatocytes are defined by the number of nuclei per cell (cellular ploidy) as well as the DNA content of each nucleus (nuclear ploidy). 21In the liver, physiological polyploidisation starts from weaning and increases with age, 22 whereas pathological polyploidisation is observed in many chronic liver diseases. 23Various mechanisms promote polyploidisation. 24Cell fusion occurs during viral infections or through receptor-ligand interactions.Cytokinesis failure results in the genesis of binucleated cells.Endoreplication encompasses endocycling (alternation of G and S phases) and endomitosis with mitotic slippage (abortion of mitosis by skipping metaphase or anaphase), 24 and thus is a cell cycledependent process.Interestingly, Mcl-1 has been shown to interact with various regulators of the cell cycle inducing divergent outcomes. 17However, these roles have been studied only in vitro.Two-thirds partial hepatectomy (PHx), extensively used in rodents to study liver regeneration in which hepatocytes synchronously re-enter cell cycle, represents a powerful tool to investigate the effect of Mcl-1 deficiency on cell cycle progression in vivo.
How ploidy and cell cycle contribute to carcinogenesis is a field of deep investigation.6][27] The 'Catalogue Of Somatic Mutations In Cancer' (COSMIC) lists many signatures identified across the spectrum of human cancers, some of which could be attributed to precise molecular aetiologies, 28 while molecular origins of many signatures remain unknown.The genomic alterations driving HCC in the different mouse models should be more systematically compared with those found in human tumours to further link mutational signatures with molecular origins. 29he unique properties of the liver regarding ploidy dynamic changes and synchronised proliferative capacity following PHx along with the spontaneous development of HCC upon Mcl-1 deficiency led us to investigate the effects of Mcl-1 deficiency on ploidy and cell cycle in murine livers in vivo, exploring possible implications for HCC.In addition, we aimed to characterise the mutational signatures of liver tumours arising from Mcl-1 deficiency and compare them with human signatures to potentially link them with underlying mechanisms.
Here, we show that Mcl-1 deficiency in young mouse liver leads to enrichment of mononuclear polyploid hepatocytes and to high rates of mitotic errors with chromosome instability such as spindle asymmetry and aberrant chromosomal segregation along with a prolonged spindle assembly checkpoint (SAC) activation signature.Mcl-1 Dhep tumours in old mice showed a high percentage of chromosomal instability and altered ploidy profile.The mutational signatures of Mcl-1 Dhep tumours were consistent among all samples and the dominant signature of currently unknown aetiology could potentially reflect mitotic issues.was done and genotyped as previously described. 6Briefly, a conditional Mcl-1 allele was generated by targeting loxP sites upstream of the ATG start codon and between exons 1 and 2. 30 The region within the two flow sites is lost and the gene is pasted back together again without this region.Mice of approximately 3 weeks of age were fed with a vitamin E-supplemented diet during 4 weeks (thus up to when the mice reached 2 months old). 8rtial hepatectomy Two-month-old male mice received food and water ad libitum before surgery.Mice were anaesthetised by inhalation of isoflurane (2%).Two-thirds PHx was performed between 9 am and 12 am.Three liver lobes, including the gallbladder, were removed.Before and after surgery, mice were treated with analgesia.Mice were euthanised by CO 2 inhalation and the regenerating liver was harvested at different time points after PHx.

Statistical analysis
Statistical analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).Data are presented as mean ± SD or mean ± SEM and were analysed by ANOVA.Analysis of two samples was performed with the Student t test.Statistical significance is indicated as follows: ****p <0.0001; ***p <0.001; **p <0.01; *p <0.05; and n.s., not significant.
For further details regarding the materials and methods used, please refer to the CTAT table and Supplementary information.

Results
Mcl-1 deficiency leads to a lower proportion of binucleated hepatocytes concomitant with a higher proportion of mononuclear polyploid nuclei in livers of young mice In Mcl-1 Dhep livers, cell density was decreased and hepatocytes exhibited increased cell size (Fig. 1A and B).To determine if cellular ploidy (number of nuclei per hepatocyte) and/or nuclear ploidy (ploidy of each nucleus) were altered, we analysed the hepatocyte ploidy profile using whole-slide imaging approaches by labelling nuclear (Hoechst) and plasma membrane (b-catenin) compartments (Fig. 1C and Fig. S1A).Regarding cellular ploidy, the binuclear fraction was lower in Mcl-1 Dhep than in WT livers at 2 months old (Fig. 1D).Regarding nuclear ploidy, the mononuclear diploid (2n) hepatocyte nuclei were substantially lower in Mcl-1 Dhep than in WT livers, whereas the proportion of mononuclear tetraploid nuclei was similar (Fig. 1E).Strikingly, Mcl-1 Dhep livers were enriched in highly polyploid nuclei (> − 8n) (15.9 ± 5.7%) which were less frequently observed in WT livers (4.9 ± 2.8%) (Fig. 1E).The data show that Mcl-1 deficiency leads to a higher proportion of enlarged mononuclear polyploid hepatocytes in livers of young mice.
Mononuclear polyploid nuclei accumulate in livers deficient for Mcl-1 independently of apoptotic activity, age, or oxidative stress levels We previously showed that hepatocyte-specific deletion of Mcl-1 triggers apoptosis and compensative proliferation in livers of 2month-old mice (Fig. 2A) with higher hepatic apoptotic levels at 2 months correlating with HCC development at 12 months. 8To evaluate the possible link between polyploidy and increased apoptosis in our model, we analysed the nuclear ploidy profile in Mcl-1 Dhep livers displaying low vs. high serum alanine transaminase (ALT) levels.We found no differences in nuclear ploidy as a function of apoptosis level (Fig. 2B).Of note, as in controls, highly polyploid hepatocytes were distributed evenly throughout all the zones of the lobule regardless of ALT levels (Fig. S1B).As 2 months of age might be too early to observe apoptotic-associated differences, we analysed nuclear ploidy in 12-month-old mice, but still observed no ploidy profile differences in Mcl-1 Dhep livers between high and low ALT levels at 2 months (Fig. 2C).To exclude that the reduction in 2n nuclei and amplification of 8n nuclei contingent in Mcl-1 Dhep livers reflected simply the proliferative activity of Mcl-1 Dhep livers, we assessed the percentage of phospho-histone H3 (pHH3)-positive nuclei (marker of cells in G2/M phase) in each nuclear ploidy category.Only a small fraction of 4n and 8n nuclei were pHH3+ in Mcl-1 Dhep livers (2% and <8%, respectively), independently of the apoptotic levels, indicating that the apparent tetraploid and octaploid nuclei were not nuclei in G2/M, thus unlikely to reflect increased proliferative activity in Mcl-1 Dhep livers at 2 and 12 months (Fig. S2A-C).
Oxidative stress was shown to promote the appearance of highly polyploid cells in fatty liver murine settings, and antioxidant-treated hepatocytes returned to physiological states of ploidy. 31As shown previously, Mcl-1 Dhep livers of mice on a chow diet exhibited 8-hydroxy-2 0 -deoxyguanosine (8-OHdG)positive hepatocytes, indicating oxidative stress, not observed anymore when mice were treated with antioxidant (vitamin E). 8 Mcl-1 Dhep mice of approximately 3 weeks of age treated for 4 weeks with normal or antioxidant diet showed comparable levels of DNA damage as assessed by ɣH2AX (Fig. S2D).In WT livers, antioxidant treatment increased 2n nuclei and decreased 4n nuclei proportion (Fig. 2D).By contrast, Mcl-1 Dhep mice treated 4 weeks with this antioxidant diet were enriched in mononuclear polyploid nuclei similarly to untreated Mcl-1 Dhep mice (Fig. 2D).
Overall, these data indicate that polyploid hepatocytes accumulate in Mcl-1 Dhep livers independently of the apoptotic levels and are not transient G2/M hepatocytes.Although antioxidant treatment altered the ploidy profile of WT livers, the data showed that oxidative stress does not contribute to the accumulation of polyploid hepatocytes in livers deficient in Mcl-1.
Mcl-1 deficiency leads to prolonged spindle assembly checkpoint activation and high rates of mitotic errors including abnormal chromosomal segregation We then aimed to investigate which mechanism(s) lead to accumulation of polyploid mononuclear hepatocytes in Mcl-1 Dhep liver in young mice.The key event of endocycling is the inhibition of entry into mitosis. 24pHH3 staining clearly showed Mcl-1 Dhep hepatocytes in mitosis, disfavouring endocycling as the main mechanism leading to nuclear polyploidisation (Fig. 3A).Endomitosis stems for issues arising during mitosis, such as prolonged SAC activation. 24We subjected 2-month-old Mcl-1 Dhep and WT mice to two-thirds PHx.PHx triggers synchronous hyperproliferation of hepatocytes of the remnant liver with a peak of proliferation at 48 h and a restored liver mass 7 days post-surgery (Fig. 3B).In Mcl-1 Dhep livers, the regenerated (7 days) to resected (0 h) liver weight ratio was significantly higher (Fig. 3C  Consistently, pHH3 labelling (G2/M marker) showed a similar mitotic index at 48 h post-PHx (Fig. 3G-I).However, in contrast to WT, some Mcl-1-deficient hepatocytes accumulated in the G2/M phase already at 24 h post-PHx.Moreover, although the mitotic index was similar (Fig. 3I) at 48 h post-PHx, the metaphase-to-telophase ratio had a tendency to be higher in Mcl-1 Dhep hepatocytes (Fig. 3J).Problems in metaphasetelophase transition have been linked to endomitosis, in which prolonged SAC activation in metaphase bypasses mitosis, known as mitotic slippage.Gene set enrichment analysis (GSEA) in steady-state livers of 2-month-old mice showed an association between genes upregulated upon Mcl-1 deletion and a mitotic SAC gene signature as well as a prometaphase signature (Fig. 4A   and B).Transcriptomic analysis from those livers revealed that among the most differentially expressed genes in Mcl-1 Dhep livers were Cdc20, required for microtubule process, the mitotic kinases Plk1, Aurora kinase A and B as well as Kif2c, encoding a microtubule protein kinesin (Fig. 4C), that is, all genes involved in mitotic regulation.We thus investigated the formation and polarisation of the microtubule mitotic spindles by assessing the morphology of mitotic figures in Mcl-1 Dhep hepatocytes in livers of 2-month-old mice using pHH3 staining.Expectedly, mitotic figures were rare in WT livers but all exhibited normal, symmetric, and bipolar metaphase plates or normal prophase nucleus (Fig. 4D).By contrast, in Mcl-1 Dhep livers, in addition to normal mitoses showing symmetrical and bipolar metaphase plate and anaphase (Fig. 4E), aberrant mitotic figures were frequently observed, including spindle multipolarity, spindle asymmetry, and abnormal spindle geometry with metaphase plate asymmetry (Fig. 4F).SAC ensures that chromosomes segregate correctly during cell division. 32Upon spindle damage, cells become transiently arrested at the metaphase-telophase transition and can exit mitosis without proper segregation of sister chromatids.Abnormal chromosome position, such as anaphase bridging and chromosome lagging (Fig. 4G), as well as prophase and prometaphase nuclei with chromosome exclusion (Fig. 4H) were observed in hepatocytes lacking Mcl-1.Quantitative b-tubulin/DAPI staining showed that approximately half of the mitotic events were aberrant upon Mcl-1 deletion (Fig. 4I).
Those findings support that, in young mice, hepatocytes lacking Mcl-1 do not undergo endocycling, enter faster into mitosis, show a SAC overactivation signature, and exhibit high rates of mitotic errors with abnormal spindle figures and chromosome mis-segregation.5A).Sequencing data were processed to identify single nucleotide variations (SNVs) with substitutions, small insertions and deletions (indels) in the coding regions.The number of SNVs was high in Mcl-1 Dhep DN (except for one mouse for which four DNs were analysed) and in HCC (Fig. 5B).The heatmap of the top 50 genes with differential variant frequencies showed coherently a stronger SNV burden in HCC compared with DN (Fig. S4A).Missense mutations were prevalent with C>T substitutions mainly found in the exomes of Mcl-1 Dhep tumours as the samples were paraffin embedded (Fig. S4A).The mean SNV and indel derived CIN was 33.8% (range: 9.1-67.7%) in the Mcl-1 Dhep tumours compared with <1% in normal tissues (Fig. 5C).
We then characterised the mutational signatures of tumours arising from Mcl-1 deficiency to compare them with human COSMIC signatures, and where possible, to assign them to known molecular aetiologies.As the formalin-fixed paraffin-embedded (FFPE)-signature is highly similar to signature 30, observed in all our samples, we introduced FFPEsig, an algorithm recently developed to rectify the formalin-induced artefacts in the mutational catalogue. 33Following this correction, the singlebase substitutions (SBSs) COSMIC signatures which most accurately reconstructed the mutational profile of Mcl-1 Dhep tumours were SBS1 and SBS87, as well as SBS6, SBS11, and SBS26 in both DN and HCC (Fig. 5D).Small insertion-and-deletion (ID) signatures ID1 and ID2 were found in 20-38% of relative contribution in DN and HCC (Fig. 5E).These mutations correlate with aging and are found in almost all cancers. 26ID1 and ID2 indels usually correlate with the numbers of SBS1 substitutions, and are proposed to reflect the number of mitoses a cell has experienced.ID12, the predominant signature, found in all tumours with around 45% of relative contribution, is of unknown aetiology.
Overall, the data showed that Mcl-1 Dhep liver tumours display chromosomal instability and a characteristic mutational signature of unknown aetiology.exhibited an increase of 4n nuclei compared with Mcl-1 Dhep nontumoral tissues (Fig. S4B).Only a very small fraction of 4n nuclei were pHH3+ in Mcl-1 Dhep livers, indicating that proliferation is unlikely to account for the observed increased 4n population in Mcl-1 Dhep tumoral hepatic tissues (Fig. S4C).Thus, the ploidy profile is altered in liver tumours spontaneously developing upon Mcl-1 deficiency with reduced cellular ploidy and increased 4n contingent.

Discussion
Mcl-1 is unique within the Bcl-2 family with respect to its short half-life and acts as a rapid sensor regulating cell death and other important cellular processes. 4,15,17,34Many studies have investigated the multiple functions and complex regulation of Mcl-1.However, its non-apoptotic roles await in vivo characterisation to develop successful Mcl-1 inhibitors for cancer therapy.

Effects of Mcl-1 deficiency on nuclear ploidy in the murine liver in vivo
Our study shows that Mcl-1 influences hepatocyte ploidy.Mcl-1 deficiency leads to less binucleated and more mononucleated polyploid contingent in the livers of young mice.This is consistent with a previous observation of enlarged cells and reduced binucleation upon Mcl-1 loss obtained with a different knockout strategy 35 owing to unknown mechanisms.Here, we demonstrated that polyploid nuclei accumulate in Mcl-1 deficient livers independently of apoptotic and proliferative levels.Accumulation of polyploid nuclei has been observed in other chronic liver disease mouse models and was attributed for fatty liver diseases to oxidative stress and associated DNA damage blocking G2/M. 30,36Here we showed that polyploid nuclei accumulate independently of oxidative stress levels in liver lacking Mcl-1 as antioxidant treatment did not rescue the nuclear profile in those livers.In addition, we found an overactivated SAC signature in livers deficient for Mcl-1.Cells that cannot satisfy the SAC are delayed in mitosis 37 and then may bypass metaphase.No differences were observed in the mitotic index 48 h post-PHx but the metaphase-to-telophase ratio had a tendency to be higher upon Mcl-1 loss.We can hypothesise that some hepatocytes lacking Mcl-1 undergo endomitosis via mitotic slippage inducing nuclear polyploidy in the next G1 phase.A slow degradation of cyclin B with simultaneously active SAC and inactive anaphase promoting complex (APC/C) complex is required for mitotic slippage to occur in humans.Further studies are required to evaluate the role of these mitotic actors in an Mcl-1-deficient hepatic setting.

Effects of Mcl-1 deficiency on cell cycle progression in the murine liver in vivo
In untreated cells in vitro, Mcl-1 protein is cell-cycle regulated with a peak in S-G2 phases both in nuclei and mitochondria. 13,39pon mild DNA damage, Mcl-1 is rapidly upregulated (in both mitochondria and nucleus) to antagonise apoptosis during the arrested cell cycle to provide time for DNA damage repair. 10,12,13hat is the impact of Mcl-1 deficiency on the cell cycle?Studies in vitro showed no impact on cell cycle progression upon Mcl-1 deletion or inhibition in untreated cells; however, Mcl-1deficient cells were blocked in G2 after irradiation 11,39 and did not show Chk1 phosphorylation and no proper G2/M checkpoint allowing correct DNA repair in response to DNA damage upon genotoxic stress.kinetochores and incorrect alignment of chromosomes on the metaphase plate prompt SAC activation and cell cycle arrest in metaphase, thereby preventing the chromosomal missegregation.However, we found that 2-month-old Mcl-1 Dhep livers displayed abnormal anaphases with lagging chromosomes, bridging chromatin (anaphase bridges) and prophase nuclei with chromosome exclusion.Mitotic asymmetry and abnormal spindle geometry during metaphase in Mcl-1 Dhep livers might induce abnormal chromosome segregation during anaphase, suggesting that Mcl-1 could be an element of the MCC.Some metaphase mitotic defects trigger less SAC activation.For instance, merotelic attachments of chromosomes, in which a single kinetochore is bound to microtubules emanating from opposite spindle poles, are poorly detected by SAC and may not be corrected, leading to lagging chromosomes during anaphase as observed here. 42mportantly, merotely is observed at high frequency during polyploid cell division because of the presence of supernumerary centrosomes and is increasingly recognised as an important mechanism contributing to CIN in polyploid cells. 43Further studies are required to clarify whether Mcl-1 has a direct role in sensing/correcting mitotic errors and/or in triggering SAC activation.
Mcl-1 deficiency leading to nuclear polyploidy and mitotic errors: implications for HCC The functional role of polyploidy in the liver is still poorly understood, especially regarding implication in HCC development.5][46][47][48] The ploidy fates seem to depend on the injury and consequent mutational context.High polyploid status seems protective against carcinogenesis except when the p53 pathway is disrupted, in which case it becomes promotive toward oncogenesis. 24A study showed that the diploid compared with polyploid state is more susceptible to tumour suppressor loss but similarly susceptible to MYC oncogene activation, indicating that polyploidy differently protects the liver from distinct genomic aberrations. 49In humans, mononuclear ploidy spectrum varies between HCC with different molecular features. 20In Mcl-1 Dhep tumours, Hras and Kras proto-oncogenes were not mutated and Ctnnb1, Apc, Braf, Egfr, and Pten were not frequently mutated (Fig. S4A).Brca2 was mutated in the five tumours arising from the same liver but not in the other livers.Thus Mcl-1 Dhep neoplasms did not carry recurrent mutations in specific genes.This is coherent with previous data showing that Mcl-1 Dhep tumours are heterogeneous with regard to morphology and immunohistochemistry and that p53 pathway is not a key player in this model. 7The homologous recombination deficiency (HRD) score was between 2 and 21, with only three tumours above 10 (data not shown) not favouring HRD as the main mechanism underlying HCC in Mcl-1 Dhep livers and further confirming the molecular heterogeneity of the Mcl-1 Dhep HCC model.
Two studies also showed that polyploid hepatocytes can divide without mitotic errors or chromosome mis-segregation upon chronic liver damage. 45,50The mitotic defects observed here could represent the consequences of cumulative DNA replication stress from the previous S phase impairing spindle organisation.Under-replicated DNA attributable to replication stress persisting in mitosis have been shown to hamper chromosome segregation. 51,52Cells entering mitosis with underreplicated DNA activate a repair mechanism known as mitotic DNA synthesis (MiDAS).During mitosis, tightly regulated pathways operate to limit the deleterious consequences of replicative stress, 53 to prevent structural and numerical chromosomal aberrations.Interestingly, although Mcl-1 Dhep tumours are heterogeneous at multiple levels, the dominant ID mutational signature found in all the 27 lesions analysed (from a total of 15 livers), is currently of unknown aetiology.As MiDAS is mechanistically and genetically similar to break-induced replication, we can hypothesise that specific mutagenic events are associated with it and might be increased during chronic proliferation as in Mcl-1 Dhep livers.MiDAS contribution to genomic mutations is still not yet understood; specifically it is not clear which SBS or ID signatures might be associated.Hence it would be potentially insightful to evaluate if Mcl-1 mutational signatures within liver tumours are linked with mitotic issues, by taking advantage notably of MiDAS sequencing approaches recently performed on the human genome. 54n conclusion, our study identified previously undescribed non-apoptotic effects of Mcl-1 deficiency on nuclear ploidy, mitosis regulation, and chromosomal segregation in adult mouse hepatocytes in vivo.In addition, the particular mutational signature of Mcl-1 Dhep hepatocellular tumours might reflect mitotic issues and deserves further investigation.Although it is not yet determined whether the effects of Mcl-1 deficiency are directly attributable to mitotic regulation or are indirect, that is, secondary to increased replication stress in a hyperproliferative environment, our results have potentially important implications for the development of Mcl-1 inhibitors as HCC therapeutics.
Performed the ploidy analysis: PC, CD.Performed the WES bioinformatic analysis: KU, PL.Performed the GSEA analysis: YB, LKC.Wrote the manuscript: LAC with input from all co-authors.(Immuno-)histological images were scanned using a SCN 400 slide scanner (Leica).

Image acquisition and analysis
In situ nuclear ploidy analysis of hepatocytes was performed as previously described [1] on whole-slide images (WSI) of β-catenin/Hoechst or pHH3/Hoechst-stained mouse liver tissue sections.Freshly stained slides were digitized using an Axio Scan.Z1 slide scanner coupled with a Colibri 7 multicolor LED light source fluorescent module (ZEISS) at 20x magnification (Plan-Apochromat 20x/0.8M27 objective) and scaling of 0.325µm x 0.325µm per pixel.
For cellular ploidy, mononuclear and binuclear fractions were quantified on 10 random highpower fields on scans of β-catenin/Hoechst stained liver sections.Hepatocyte nuclear ploidy was inferred from Hoechst staining and nuclear area with a specific method based on stereological image analysis and implemented in ZEN (ZEISS) and Image J software.Briefly, after Hoechst fluorescence intensity adjustment and automatic segmentation of all nuclei on the WSI, the density function of the nuclear area was plotted, and a Gaussian mixture model was fit to the multimodal distribution.Secondly, area thresholds were graphically determined to separate 2n (>30-60 μm2), 4n (>60-90 μm2) and ≥8n (>90-500 μm2) nucleus populations.
The proportion of hepatocyte nuclei in each nuclear ploidy category was finally obtained by reporting the result to the total number of hepatocyte nuclei detected on the WSI.Nonhepatocyte nuclei from non-parenchymal cells (NPCs) were filtered out based on circularity (close to 1 for a hepatocyte nucleus) and nuclear area.Circularity thresholds were set at 0.8 in non-tumoral liver tissue and 0.7 in liver neoplasms to account for higher degrees of nuclear pleomorphism.Nuclei with a measured area <30 μm2 or >500 μm2 were also excluded from the analysis (NPC nuclei or incorrectly segmented nuclei with fusion or fragmentation).Three to five liver lobes were analyzed for each mouse to account for potential inter-lobar heterogeneity.On average 80,000 ± 28,000 hepatocyte nuclei were analyzed per mouse.For nuclear ploidy map reconstruction (Figure S1), a second Image J macro was used to record the coordinates of nuclei (centroid position) which were subsequently mapped and color-coded according to their nuclear ploidy: NPC nuclei in blue, 2n hepatocyte nuclei in purple, 4n hepatocyte nuclei in green and ≥8n hepatocyte nuclei in red.To determine the proportion of G2/M hepatocyte nuclei in each nuclear ploidy category, pHH3 positivity was detected on WSI after manual thresholding.The number of positive nuclei was then reported to the total number G2/M hepatocyte nuclei per nuclear ploidy category (pHH3+ 2n, 4n and ≥8n nuclei).Representative images were captured using a Nikon DS-Ri1 microscope camera and NIS-Elements Br software.
and S3A-C).Ki67+ cells appeared already at 24 h post-PHx in Mcl-1 Dhep compared with WT (Fig. 3D), whereas Ki67+ cells were virtually absent in both WT and Mcl-1 Dhep livers at 6 h post-PHx (Fig. S3D) suggesting that the basal proliferation in Mcl-1 Dhep is not the main reason for faster regeneration.When assessing hepatocyte replication by monitoring bromodeoxyuridine (BrdU) incorporation, BrdU was detected already after 24 h in Mcl-1 Dhep livers and peaked in both WT and Mcl-1 Dhep mice 48 h post-PHx (Fig. 3E and F).

Fig. 4 .
Fig. 4. Mcl-1 deficiency leads to prolonged SAC activation, mitotic errors and chromosome mis-segregation in steady-state livers of 2-month-old mice.(A, B) GSEA comparing all differentially regulated genes from livers of 2-month-old Mcl-1 Dhep mice with various gene sets.(C) Table of the 10 most enriched genes involved in the regulation of metaphase obtained by GSEA analysis of 2-months-old WT vs. Mcl-1 Dhep mice.(D).Representative images of Hoechst and pHH3 staining of livers of 2-month-old WT mice displaying normal prophase nucleus and symmetric bipolar metaphase plate.Representative images of Mcl-1 Dhep mice displaying (E) normal and (F) abnormal mitotic figures with spindle multipolarity and asymmetry, (G) chromosome exclusion as well as (H) lagging and bridging chromosomes.(I) Percentage of aberrant mitotic figures relative to normal mitotic figures in Mcl-1 Dhep livers.GSEA, gene set enrichment analysis; Mcl-1, myeloid cell leukaemia sequence 1; pHH3, phospho-histone H3; SAC, spindle assembly checkpoint; WT, wild type.

Fig. S2 .
Fig. S2.Mononuclear polyploidy nuclei accumulating in livers lacking Mcl-1 are not transient G2 nuclei.A. Representative images of pHH3/Hoechst staining and ploidy map showing the 2n, 4n and 8n nuclear contingents.B,C.Percentage of pHH3 positive hepatocytes in mononucleated 2n, 4n and 8n contingent in WT, Mcl-1 Δhep displaying low or high ATL levels of 2 and 12 months old mice (n=4-8 per group).D. Percentage of ɣH2AX positive hepatocytes in livers of 2 months old WT and Mcl-1 Δhep mice treated or not with vitamin E for 4 weeks (n=4-6 per group).Statistical test: one-way ANOVA test with Tukey's multiple comparisons test when significant.n.s.: not significant.*p < 0.05, ***p-value ≤ 0.001.Data are expressed as mean ± SEM.

Fig. S4 .
Fig. S4.Not recurrently mutated genes are observed in Mcl-1 Δhep tumors of 12 months old mice.A. Plot of the 50 differential genes in DN and HCC Mcl-1 Δhep samples and mutations in common oncogenes per individual tumor.
After pHH3/Hoechst fluorescence immunostaining, liver tissue sections were analyzed directly under a Nikon Eclipse E600 upright microscope equipped with Plan Fluor 20x/0.75 and 40x/1.30Oil objectives and 10x/22 oculars.pHH3+ hepatocyte nuclei were sought at 200x magnification, and their morphology analyzed at 400x magnification with a combination of pHH3 and Hoechst staining to better determine the mitotic stage and chromosome arrangement.G2 nuclei were discriminated from M (Mitotic) nuclei based on the nuclear morphology (presence of nuclear envelope, intermediate state of chromatin compaction and absence of chromosome individualization) and the pattern of pHH3 positivity (discrete nuclear foci overlapping heterochromatin regions).Aberrant mitotic figures (AMF) were defined as mitoses whose morphology deviates substantially from normalcy for the corresponding mitotic stage (generally bipolar and symmetrical metaphase and symmetrical anaphase with equal partitioning of chromosome)[2].They broadly include mitotic/polar asymmetry and abnormal segregation of chromosomes in anaphase.The following AMF were considered in the present study: asymmetrical bipolar mitosis with unequal distribution of chromosomes along the metaphase plate (metaphase plate asymmetry) or unequal repartition of chromosomes in anaphase, multipolar mitosis with more than 2 spindle poles, mitosis with spindle asymmetry or abnormal spindle geometry and mitosis with abnormal chromosome segregation in anaphase (lagging chromosome and anaphase bridge).pHH3+ NPC nuclei were not considered and discriminated based on nuclear size, morphology, and localization relative to hepatocyte plates.
All animal experiments conformed to the relevant regulatory standards and were approved by the Swiss Veterinary Office (134/2014, 217/2012, 63/2011, 03/2015 Zurich, ZH104/19).Animals were maintained under pathogen-free conditions and experiments were performed in accordance with the guidelines of the Swiss Animal Protection Law, Veterinary Office, Canton Zurich.Generation of mice with hepatocyte-specific Mcl-1 knockout (homozygous: Mcl-1 flox/flox -AlbCre [referred to as Mcl-1 Dhep ] and control littermates: Mcl-1 wt/wt [referred to as WT]) Dhephepatocytes.Hyperproliferative liver is a clinically relevant situation, not only after liver resection in patients with HCC.Hence further studies are needed to understand the effect of Mcl-1 loss in this context, which may be conductive to successful clinical application of Mcl-1 inhibitors.
41The failure to undergo complete mitosis after DNA damage coupled to defective checkpoints, called mitotic catastrophe,40has also been shown to lead to polyploidy with DNA damage.41Here, weobserved that Mcl-1 Dhep hepatocytes enter faster into S, G2, and M phases in vivo but accumulate similarly 48 h post-PHx.Chk1 This large proportion of mitotic errors is consistent with the numerous chromosomal deletions and amplifications revealed by array-comparative genomic hybridization analysis of Mcl-1 Dhep livers. 7The transcriptomic signature of overactive SAC identified in steady-state livers of 2-month-old Mcl-1 Dhep mice indicate that those mitotic aberrations are detected and triggered mitotic checkpoint complex (MCC) assembly.Unattached