Introduction

Epigenetic alterations which are reversible, yet heritable, changes in histone proteins and the DNA are an integral component of tumor progression1. Next-generation sequencing allowed the identification of frequent mutations in epigenetic regulators various sporadic cancers. Among those, the mixed-lineage leukemia (MLL) family of histone methyltransferases, which methylate the histone 3 tail at lysine 4 (H3K4)2 as part of the Complex Proteins Associated with Set1 (COMPASS) complex3 stand out both with respect to mutation frequency but also the wide spectrum of neoplasias involved4,5,6. MLL3 (also known as KMT2C) is among the most frequently mutated proteins in urothelial carcinoma. The majority of mutations lead to truncated proteins, indicating a tumor suppressor role7,8. The tumor suppressor role of KMT2C in urothelial tumorigenesis is further supported by experiments in mouse models of cancer9, while more recently it was shown that genetic inactivation of KMT2C or KMT2D in vivo primes mouse urothelium for neoplastic transformation10.

Bladder cancer is among the most common solid malignancies in humans11, with the majority of cases being urothelial carcinomas. Of those, 75% are diagnosed as superficial non-muscle invasive cases while the remaining 25% are already muscle-invasive or metastatic at the time of diagnosis12. KMT2C mutations are common in both types8,13,14. While non-muscle-invasive bladder cancer (NMIBC) often progresses to muscle invasive disease (MIBC), the genetic events driving progression have not been fully understood, while patient stratification relies mostly on clinicopathological characteristics. Despite recent progress15, there is a strong unmet need for molecular stratification.

Eukaryotic cells have evolved robust cell cycle checkpoint systems that allow maintenance of genome integrity in the presence of high levels of DNA damage and replication stress. The G1/S and G2/M checkpoints prevent tumor cells with unreplicated or damaged DNA from entering catastrophic mitoses16,17,18.

Here we show that low KMT2C expression levels are associated with worse prognosis in bladder cancer patients. We then utilize KMT2C knock-down (KD) bladder cancer cell lines to uncover activated pro-tumorigenic pathways and identify defective cell cycle checkpoints and activation of PDK/AKT signaling. We find that this is coupled with cell cycle checkpoint progression in the presence of DNA damage and higher frequency of aberrant mitosis. As a result, PLK1 inhibition in cells with KMT2C loss leads to mitotic catastrophe and cell senescence, which translates to significantly slower tumor growth in vivo.

Results

Reduced KMT2C expression is associated with poor survival in bladder cancer patients

In a previous study where we generated a cohort of urothelial tumors, we found that KMT2C in cancer tissue is transcriptionally downregulated in the majority (70%) of cases14. Interestingly, KMT2C levels negatively correlate with tumor stage (p < 0.001) and grade (p < 0.001) in our cohort composed of both muscle-invasive (MI) and non-muscle invasive BC (NMIBC) patients (Fig. 1a)14,19. This finding was corroborated in an independently published cohort20 (Fig. 1b). More importantly, low levels of KMT2C are associated with poorer clinical outcome, with significantly shorter overall survival (OS) in the MIBC cohort, and disease-free survival (DFS) and progression-free survival (PFS) of the NMIBC group of patients (P = 0.014, 0.037 and <0.001, respectively; Fig. 1c–e).

Fig. 1: Reduced KMT2C activity is associated with more aggressive clinical characteristics.
figure 1

a Box plots indicating relative KMT2C expression with respect to tumor grade and stage in our cohort. The correlation of KMT2C expression with tumor grade and stage was assessed by Mann-Whitney U and Kruskal-Wallis tests, respectively. For pairwise comparisons between tumor stages Mann-Whitney U test was used. ****p < 0.0001, ***p < 0.001, *p < 0.05. b Relative KMT2C expression with respect to tumor grade and stage in an independent published cohort20. The geometrical mean of log2 values from 4 different probes was used and the significance was assessed by Mann-Whitney U test. c–e Kaplan-Meier survival curves of bladder cancer patients with respect to KMT2C expression levels. “n” refers to the number of patients and “events” refers to deaths or cases of disease recurrence, depending on the plot. For all plots, median was used as the cut-off between the two groups. OS overall survival, DFS disease-free survival, PFS progression-free survival, MS Median survival. p-value calculated by log-rank test. f Box and whisker plot (2.5-97.5 percentile) showing the levels of total and phosphorylated PDK1(Ser241) in TCGA bladder cancer patients7 with WT and mutant KMT2C. Outliers beyond the end of the whiskers are also indicated. Man-Whitney test was employed for statistical analysis. **p < 0.01; ns: not significant (p > 0.05).

Full size image

To identify potential mechanisms that explain the association between reduced activity of KMT2C and disease progression, we utilized datasets from the cancer genome atlas (TCGA). Reverse phase protein array (RPPA)7 data reveal that phosphorylated PDK1 (pPDK1) is one of the top upregulated proteins in KMT2C mutant vs. wild-type tumors (Figs. 1f, S1). Phosphorylated PDK1 (at Ser241) is a well-established, critical upstream activator of the AKT/mTOR signaling cascade. This pathway is one of the most frequently hyperactivated pathways in human cancers, driving cell proliferation, growth, and survival, and could explain the worse prognosis of patients with reduced expression of KMT2C.

Loss of KMT2C activity impairs cell cycle regulation through PI3K/AKT activation

To study how KMT2C loss regulates tumor promoting pathways, we used the human bladder cell lines HTB9 and T24 and established cells expressing shRNAs against KMT2C (KD1 and KD2) or scramble control (Scr) to induce downregulation of KMT2C (Fig. 2a, b, S2A). Gene ontology (GO) analysis of significantly upregulated genes in KMT2C/KD1 cells, from a previously-published RNA-seq experiment14, identified an enrichment in signatures associated with cell cycle progression, including E2F targets and G2/M checkpoint components (Fig. 2c, Table S1). Specifically, KMT2C/KD cells show upregulation of E2F transcription factors (E2F1/2/3) and cyclins (CCND1/E1). In agreement with a previous report implicating KMT2C in promoter binding and transcription regulation of cell cycle genes21, KMT2C/KD cells show downregulation of the cyclin-dependent inhibitors CDKN1B (p27) and CDKN1C (p57) (Fig. 2d–e). These transcriptional changes lead to corresponding changes at the protein level (Figs. 2f, S2B). Additionally, KMT2C/KD cells showed upregulation of the G2/M checkpoint genes PLK1 and AURKA, which are known to promote mitosis in normal and neoplastic cells22,23,24,25, genes encoding components of the anaphase promoting complex (APC; ANAPC2, UBE2C and CDC20), centromeric region proteins (SPC25, AURKA, AURKAB and PLK1), and mitotic spindle assembly checkpoint proteins (PLK1, AURKB and CDC20) (Fig. S2C, D).

Fig. 2: Loss of KMT2C activity impairs cell cycle regulation through PI3K/AKT activation.
figure 2

a Quantitative RT-PCR on HTB9 control (Scr), KMT2C/KD1 and KMT2C/KD2 cells. b Western blots with protein extracts from HTB9 control, KMT2C/KD1 and KMT2C/KD2 cells with the indicated antibodies. c Gene ontology analysis (MSigDB Hallmark 2020) of upregulated genes in HTB9 KMT2C/KD1 cells identified by differential gene expression analysis of previously generated RNA-seq data14. d–e Heatmap based on RNA-seq data from KMT2C/KD1 and qRT-PCR from KMT2C/KD2 cells indicating the expression levels of G1/S components in control (Scr), KMT2C/KD1 and KMT2C/KD2 HTB9 cells. Western blot analysis of G1/S components (f), PDK/AKT signaling (g), and PTEN (h) in parental, control (Scr), KMT2C/KD1 and KMT2C/KD2 HTB9 cell extracts. i Bedgraphs from previously published ChIP-seq data14. indicating KMT2C (Flag-KMT2C) binding on the promoters of CDKN1B and PTEN and histone modifications H3K4me3 and H3K9ac in control (Scr) and KMT2C/KD1 HTB9 cells. j Western blot analysis of PTEN and PDK/AKT signaling using cell extracts from control and KMT2C/KD1 HTB9 cells, in which PTEN has been exogenously re-expressed. Western blot analysis against phosphorylated and total AKT1 (k) and cell cycle analysis (l) of Scr and KMT2C/KD1 and KD2 HTB9 cells following treatment with PDK1 inhibitor (30 μM) at the indicated timepoints.

Full size image

In addition to cell cycle, loss of KMT2C is associated with gene expression changes in the PI3K/AKT axis (Fig. 2c). Western blot analysis of control and KMT2C/KD cells indicates that loss of KMT2C activity leads to strong elevation of phosphorylated PDK1 and AKT1 levels, indicating activation of this major pro-survival pathway (Figs. 2g, S2E). To gain insight on the cause of PDK1/AKT1 activation, we assessed protein levels of PTEN by western blot analysis and found that upon KMT2C knockdown in HTB9 and T24 cells, PTEN levels are diminished (Figs. 2h, S2F).

To investigate whether KMT2C plays a direct role in regulation of cell cycle genes we analyzed previously generated ChIP-seq experiments in control HTB9 (Scr), KMT2C/KD1 cells, and KMT2C/KD1 cells complemented with a Flag-KMT2C fusion which partially restores KMT2C activity14. KMT2C itself is recruited at the proximal promoters of CDKN1B and PTEN, but not CDKN1C (Figs. 2i, S2G), suggesting direct and indirect roles of KMT2C in epigenetic and transcriptional regulation of these genes. In addition, loss of KMT2C leads to a marked reduction of histone marks associated with positive regulation of gene expression at the CDKN1B and PTEN promoter, specifically H3K4me3 and H3K9ac (Fig. 2i). Notably, putative enhancer regions within 100kb from the transcription start site (TSS) of CDKN1B and PTEN show lower KMT2C binding compared to promoters, (Figs. S2H), which suggests that KMT2C binding on respective promoters is more critical for transcription regulation.

Together these results support a role for KMT2C in regulation of cell cycle transcriptional programs, both directly through CDKN1B and indirectly through the PTEN/PDK/AKT axis. In agreement with this, exogenous re-expression of PTEN in a Doxycycline-inducible manner leads to repression of the PI3K pathway as indicated by phosphorylated levels of PDK1 and AKT1 (Figs. 2j, S2I). Additionally, cell cycle analysis of HTB9 cells treated with low concentrations (30 μM) of the PDK inhibitor GSK2334470 (Fig. 2k) indicated that, unlike control cells, KMT2C/KD1 and KD2 cells fail to progress through the G1/S checkpoint (Fig. 2l), implying a severe addiction of KMT2C/KD cells to PI3K/PDK1/AKT axis for cell cycle progression. Treatment with the clinically-approved PIK3CA inhibitor Pictilisib (Fig. S2J) confirmed this dependence, in agreement with the role of PI3K/PDK1/AKT in favoring proliferation at the expense of E2F-driven apoptosis26,27,28.

These in vitro findings agree with transcriptomics analyses from the TCGA bladder cancer dataset. Specifically, we identify a strong correlation (Pearson r=0.485, p<0.0001) between KMT2C and CDKN1B expression (Fig. S3A), and higher E2F1 expression levels in tumors with low expression of KMT2C (Fig. S3B). The latter finding is more pronounced in the context of wild-type RB1 (Fig. S3C), likely because RB1 loss leads to high protein and transcript levels of E2F129. Finally, a strong inverse correlation between KMT2C and a G2/M signature was identified (Fig. S3D, E). Interestingly, the E2F family of transcription factors has been implicated in PLK1 gene transcriptional activation30,31. To this direction, ENCODE ChIP-seq data32 indicate that most genes in this G2/M signature are indeed direct targets of E2F1 (Fig. S3F). Altogether, these data indicate that KMT2C loss leads to defective cell cycle regulation and activation of the oncogenic PI3K/PDK/AKT axis.

KMT2C-deficient cells progress through the cell cycle with high DNA damage

We previously showed14 that KMT2C loss is associated with high levels of genomic instability. To study the dynamics of progression through the G1/S and G2/M checkpoints in relation to DNA damage induced by KMT2C loss, we employed the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system33, which allows visualization of cells in the different phases of the cell cycle. Cells with high numbers of γH2AX foci in the G1 phase (labeled red by CDT1) and the late S/G2 phases (labeled green by Geminin) are more frequent in the KMT2C/KD cells (Fig. 3a, b). Much of this damage is sustained in the S phase as indicated by the immunofluorescence detection of γH2AX foci in PCNA-positive cells (Fig. 3c). Despite the high DNA damage, EdU-positive DNA replicating KMT2C/KD1 cells display lower number of MRE11 foci (Fig. 3d), in agreement with previous reports showing that loss of KMT2C, which recruits MRE11 at stalled replication forks34, and protects replication forks from degradation in homologous recombination deficient (HRD) cells.

Fig. 3: KMT2C-deficient cells progress through the cell cycle with high DNA damage.
figure 3

Immunofluorescence detection of γH2AX levels in control and KMT2C/KD1 HTB9 cells expressing the Cdt1 (a) and the geminin (b) FUCCI reporters. A quantitative analysis of the number of γH2AX foci in Cdt1 (left) and geminin (right) positive cells between control and KMT2C/KD1 HTB9 and T24 cells is indicated. c Immunofluorescence detection of γ-H2AX in control and KMT2C/KD1 cells (HTB9 and T24) expressing mTagRFP-PCNA and quantification of the percentage of PCNA positive cells with more than 10 γ-H2AX foci in these cells. d Co-localization of EdU (red) and MRE11 (yellow) in control and KMT2C/KD1 cells (HTB9 and T24) and quantification of the percentage of γ-H2AX positive cells (Y-axis) with the indicated numbers of MRE11 foci (X-axis) in these cell lines. Schematic representation of the G2 checkpoint inhibition assay (e) and mitotic index values (f) in control and KMT2C/KD1 HTB9 and T24 cells that were subjected to this assay. Un designates untreated cells. IR designates irradiated cells. For each condition at least 700 nuclei were analyzed. g Immunofluorescence detection of γH2AX in metaphases and anaphases from control and KMT2C/KD1 mitotic cells. A comparative analysis of the number of γH2AX positive mitoses between control and KMT2C/KD1 HTB9 and T24 cells is indicated by the bar graph. For all quantitative analyses, data are presented as mean ± standard error of the mean (SEM) from 3 independent experiments and statistical significance was determined by unpaired Student’s t-test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns: not significant. A total of 75, 91, 75 and 93 metaphases/anaphases were analyzed from HTB9/Scr, HTB9/KD1, T24/Scr and T24/KD1, respectively. Scale bars indicate 5 μm.

Full size image

To investigate the effect of G2 component overexpression (Figs. 2c, S2C, D) in cell cycle progression, we used a G2 checkpoint inhibition assay (Fig. 3e). Exposure of cells to ionizing radiation, a known inducer of DSBs35, causes G2 arrest. Colcemid treatment shortly after irradiation traps early G2-irradiated cells that have escaped G2 arrest and progressed to mitosis, in metaphase35. Through this assay we observed that KMT2C/KD1 cells have a higher mitotic index (Fig. 3f) and higher frequency of mitotic cells with DSBs (Fig. 3f, g). Together, these results indicate that KMT2C loss is associated with progression of the cell cycle with high levels of unresolved DNA damage.

KMT2C loss leads to mitotic defects

Further characterization of the effects of KMT2C loss on mitosis by live cell imaging indicated that metaphase-to-anaphase progression is much slower in KMT2C/KD cells compared to control cells (Fig. 4a), with the majority (90%) of mitoses requiring over 120 min to progress (Fig. 4b, c). Delayed mitotic exit is a hallmark of mitotic stress characterizing tumors with high replication stress and genomic instability36,37,38. In agreement with this, KMT2C/KD1 cells display a high frequency (38% vs. 8%, p < 0.001) of anaphase errors such as chromosome bridges and lagging chromosomes (Fig. 4d), as well as centrosome amplification (Fig. 4e).

Fig. 4: KMT2C loss leads to mitotic defects.
figure 4

a Boxplot indicating the time required for metaphase-to-anaphase progression in control and KMT2C/KD1 HTB9 cells. Mitotic progression data for each cell line were acquired by automated time‑lapse microscopy of control (n = 32) and KMT2C/KD1 HTB9 cells (n = 32) expressing mCherry-tagged Histone H2B. The time point t = 0 was defined as the time point at which a perfect metaphase plate was observed and the time point corresponding to anaphase onset was calculated from the first frame at which chromosome segregation was visible. p-values were calculated by a Mann-Whitney test. b Bargraph indicating the percentages of control and KMT2C/KD1 HTB9 mitotic cells that either progressed from metaphase plate to anaphase onset within a time frame of 120 min or failed to so (not progressed). c Time-lapse microscopy of indicative mitosis of control HTB9 cells showing anaphase onset in 65–70 min and KMT2C/KD1 HTB9 cells that progressed to anaphase with a delay (235 min). d Representative images and quantification of defective anaphases in control and KMT2C/KD HTB9 cells. Mitotic cells were fixed and stained to reveal DAPI (blue) and pH3 Ser10 (green). Mitoses with chromosome bridges and lagging chromosomes were included in the analysis. Average of 3 experiments are shown. A total of 412 and 372 mitoses were analyzed for Scr and KD1 cells, respectively. Scale bar = 5 μm. e Representative images and quantification of cells with centrosome amplification of control and KMT2C/KD HTB9 and T24 cells. Cells were fixed and stained to reveal DAPI (blue) and γ-tubulin (red) and were captured with the 100X objective lens of the fluorescent microscope used. A total of 2831, 1541, 981 and 966 cells were analyzed from HTB9/Scr, HTB9/KD1, T24/Scr and T24/KD1, respectively. Scale bar = 25 μm. f Schematic outline of the experiment to assess PLK1-dependence of control and KMT2C/KD cells for mitotic entry. g Immunofluorescence detection of phosphorylated histone H3 in untreated and PLK1 treated control and KMT2C/KD HTB9 cells for the quantitative analysis of mitotic cells. DAPI is used as nuclear counterstain. Scale bar = 25 μm. h Bargraphs indicating the percentage of mitotic cells in synchronized cultures of control and KMT2C/KD1 HTB9 and T24 cells that were either treated by vehicle (DMSO) or treated by PLK1 inhibitor volasertib (1 μM). Quantification of phospho- histone H3 positive nuclei was performed at 12 h (t = 0 h) and at 16 h (t = 4 h) after thymidine block release as presented to the schematic outline (f). The Y values indicate the average from 3 experiments fold changes of the percentage of mitotic cells based on the percentage of mitotic cells at t = 0 h for each cell line. At least 500 nuclei were measured for each condition. i Representative images of (upper) and quantitative analysis (lower) of mitotic catastrophe events in PLK1 treated control and KMT2C/KD cells that express mCherry-tagged Histone H2B. Mitotic catastrophe development was evaluated by live cell imaging and analysis of nuclear morphology. Yellow circles indicate fragmented/multilobular nuclei that are characteristic of mitotic catastrophe events. For all quantitative analyses, data are presented as mean ± standard error of the mean (SEM) from 3 independent experiments and statistical significance was determined by unpaired Student’s t-test, unless indicated otherwise. ****p < 0.0001, *** p < 0.001, **p < 0.01, *p < 0.05, ns not significant. Scale bar = 5 μm.

Full size image

To investigate whether the overexpression of mitotic checkpoint components such as PLK1 in KMT2C/KD cells is important for mitotic progression and cell viability, we used the well-studied PLK1 inhibitor Volasertib (PLKi) in synchronized cultures of control and KMT2C/KD1 cells (Fig. 4f). Although mitotic entry of both control and KD cells is attenuated by PLKi treatment, KMT2C/KD1 cells fail to overcome PLK1 inhibition and progress to mitosis, as indicated by immunofluorescence detection of phosphorylated histone H3 levels (Figs. 4g, h and S4). PLK1 inhibition has been associated with mitotic defects and mitotic catastrophe events39. In agreement with such findings, a higher frequency of mitotic catastrophe (fragmented/multilobular nuclei) is observed in KD cells (Fig. 4i). Together, these results suggest that KMT2C loss confers a dependency on PLK1 for successful completion of mitosis, possibly due to high levels of mitotic stress.

PLK1 inhibition leads to increased senescence of KMT2C/KD cells

Due to the translational significance of PLK inhibition in cancer treatment, we sought to further characterize its effect in the context of KMT2C loss. Microscopical evaluation of PLKi-treated asynchronous cultures from the HTB9 and T24 bladder cancer lines indicated an increased frequency of cells with senescence-associated phenotype in KMT2C/KD cells, an observation that was confirmed by senescence-associated beta-galactosidase (SA-β-gal) activity (Figs. 5a, S5A, B). This is consistent with previous reports showing that PLK1 inhibition induces cellular senescence in human primary and cancer cells40,41. Western blot analysis on HTB9 cells showed a higher increase of p21Waf/Cip1 and p16INK4A protein levels in KD1 vs. Scr cells upon PLK1 inhibition, further corroborating the senescence phenotype (Figs. 5b, S5C, D).

Fig. 5: PLK1 inhibition leads to increased senescence of KMT2C/KD cells.
figure 5

a Representative photos of control and KMT2C/KD1 and KD2 HTB9 and T24 cells with or without PLKi treatment at two different concentrations (Un = Untreated, 10 nM, 30 nM), stained with SA-β-Gal staining solution. Graph showing the percentages of SA-β-Gal positive cells in control and KMT2C/KD1 and KD2 HTB9 cells) that have been treated or not with PLKi. In bargraphs, one-way ANOVA was used and statistically significant pairwise comparison with respective vehicle is indicated with stars on top of each column. b Western blots showing p21 and p16 levels from control and KMT2C/KD1 and KD2 HTB9 cells that have been treated with PLKi at three different concentrations (Un = Untreated, 10 nm, 30 nm). Antibody against GAPDH was used as loading control. c Tumor growth obtained from xenografts of control (top) and KMT2C/KD1 (bottom) HTB9 cells treated with vehicle or volasertib (12.5 mg/kgr, twice a week). Number of mice used: n = 7 for Scr and n = 9 for KD1. Mann-Whitney U test was used in statistical analysis of pairwise comparisons. d Immunofluorescence with antibody against p21 on tumor sections from vehicle and volasertib treated xenografts mice that were generated by control and KMT2C/KD1 HTB9 cells. e Western blot analysis and quantitation of p21 protein levels on tumor lysates from vehicle and volasertib treated xenograft mice that were generated by control and KMT2C/KD1 HTB9 cells. Student’s t test was used for statistical analysis. f Immunocytochemistry using GL-13 (Sentragor) on tumor sections from vehicle and volasertib treated xenografts mice that were generated by control and KMT2C/KD1 HTB9 cells. For all statistical analyses, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant.

Full size image

To explore the clinical implications of PLK1 inhibition, we generated in vivo xenograft models using control and KMT2C/KD1 cells. PLKi treatment indicated that KMT2C/KD1 cells are highly sensitive to PLK1 inhibition (Fig. 5c). The reduced tumor growth of KMT2C/KD1 cells can be at least in part attributed to induced senescence, as indicated by p21Waf/Cip1 immunofluorescence and western blot analysis of xenograft tumors (Figs. 5d, e, S5E). Moreover, the senescence phenotype was evaluated with the use of GL13 staining (Fig. 5f), a robust biomarker assay to detect lipofuscin granules in senescent cells42.

Altogether, our data indicate that reduced KMT2C activity leads to aberrant cell cycle progression associated with tolerance to DNA damage. However, this adaptation, which relies on cell signaling and gene expression changes, creates novel dependencies that are therapeutically exploitable.

Discussion

Here, we show that loss of KMT2C activity is associated with a bad prognosis of BLCA patients. Differential expression and protein level analysis in KMT2C knock-down cell lines indicates that loss of KMT2C activity is associated with positive regulation of the G1/S cell cycle checkpoint, and activation of the PI3K/PDK/AKT signaling pathway. Using immunostaining assays, metaphase preparations, and the FUCCI system for cell cycle analysis, we detected that KMT2C/KD cells are characterized by high amounts of DNA damage and chromosomal aberrations during G2/M and S phases of the cell cycle. As a result of this genomic instability, inhibition of the mitotic factor PLK1 in KMT2C/KD cells leads to mitotic catastrophe, cell senescence, and reduced growth in vivo.

Our ChIP sequencing experiments point to a novel, direct role for KMT2C in regulation of the CDKN1B and PTEN gene loci through binding at their promoters and loss of activating histone marks. This finding is in agreement with previous reports showing epigenetic regulation of the proximal promoter of CDKN1B by EZH2 in glioma and hepatocellular carcinoma43,44, and PTEN in various cancers45,46,47,48,49,50. Importantly, activation of the PI3K/PDK/AKT axis, e.g., by PTEN loss, has been implicated in bladder cancer development in humans7, and is associated with overexpression of E2F members, and cell proliferation triggered by E2F overexpression at the expense of apoptosis26,27,28. On the other hand, E2F1 levels positively correlate with G2/M component expression. Seemingly, E2F1 plays a central role in both G1/S and G2/M progression, at least in the context of KMT2C-low tumors. Together, transcriptional dysregulation of these genes and their downstream targets by KMT2C loss points to a pro-proliferative phenotype that could explain the association of reduced KMT2C expression and aggressive disease. This hypothesis is supported by gene expression correlation analyses in patient datasets.

While this study focuses on the effect of KMT2C loss in already neoplastic human bladder cells, reports in the literature indicate that in vivo inactivation of KMT2C in mice, although not sufficient on its own, leads to cancer development in collaboration with Tp53 or Pten loss9,10. Notably, KMT2C loss leads to extensive epigenetic changes both in bladder cancer cells and normal mouse urothelial cells. This rewiring can be associated with a more aggressive phenotype and altered therapeutic responses14 or could prime normal cells for neoplastic transformation10.

Dysregulation of cell cycle checkpoints has been previously associated with chromosomal defects51. This suggests an additional mechanism by which KMT2C promotes high levels of chromosomal instability, besides its role in homologous recombination repair14,34,52. Indeed, this downstream effect of KMT2C loss creates a dependence on PLK1, a key protein for G2/M checkpoint exit, for cell cycle progression22,23,24. In fact, PLK1 is known to promote tolerance to genotoxic stress53,54.

PLK inhibition has been associated with G2/M arrest55, mitotic catastrophe events39, and cell senescence40,41. As a result, the PLK1 inhibitor volasertib is investigated in patients with locally advanced or metastatic urothelial cancer in phase II clinical trials56. In line with this, our in vitro and in vivo pre-clinical experiments support PLK inhibition as a targeted therapy for tumors with compromised KMT2C activity, either due to mutation or reduced expression levels.

Overall, this study highlights novel mechanisms of cell cycle and genome stability regulation by KMT2C and provides a basis for future clinical studies investigating PLK inhibitors as a targeted therapy for bladder cancer patients with KMT2C loss.

Methods

Human specimens

The primary bladder cancer cohort has been described14. All patients were treated at Laiko General Hospital, Athens, Greece. Healthy adjacent tissue was obtained for 104 patients. Pathology evaluation confirmed the absence of tumor cells in the healthy specimen. All patients were treatment naïve. No inclusion/exclusion criteria, other than tissue quality upon thawing were used. This study was performed according to the ethical standards of the 1975 Declaration of Helsinki, as revised in 2008, and was approved by the ethics committees of Laiko General Hospital. Informed consent was obtained from all subjects.

Mouse experiments

Male NOD/SCID mice were purchased from the Jackson repository and bred in individually ventilated cages at the Animal House Facility of the Foundation for Biomedical Research Foundation of the Academy of Athens (Athens, Greece). All procedures were approved by the Institutional Committee on Ethics of Animal Experiments and the Greek Ministry of Agriculture (Protocol No. 893684/19-09-2022). Mice were anesthetized with isoflurane and one million (106) cells were injected in the flank of 6–8 weeks-old mice. A total of 20 mice were injected each with control (Scr) and KMT2C/KD1 cells. Treatment commenced when tumors reached a palpable size (app. 2 mm in diameter). Mice were randomly assigned to the vehicle and volasertib groups. No blinding was applied. No exclusion criterion was applied, except the lack of growth of palpable tumors at the initiation of thew treatment. Cohorts were maintained in separate but adjacent cages, and mice within each cage were injected in a random order. Volasertib was administered twice a week (D1 and D4) at 12.5 mgr/kgr mouse weight in 30% PEG400, 0.5% Tween 80, 0.5% propylene glycol vehicle for in vivo efficacy studies. Cohort size was 7 and 9 mice for control (Scr) and KMT2C/KD1 xenografts, respectively for both vehicle and volasertib, which is in agreement with the standards in the literature. Measurements were taken on days 5, 10, 15, and 20 of the treatment with caliper and calculated as V = 1/2a × b2, “a” being the largest diameter, “b” the smallest. No treated animal was excluded from the statistical analysis. Mean tumor volumes were plotted. Mann-Whitney U test was used in statistical analysis of tumor growth kinetics. At the end of the study, mice were euthanized with cervical dislocation.

BLCA TCGA data

All meta-analyses performed in this manuscript used data generated by The Cancer Genome Atlas (TCGA) research network which were retrieved from cBioPortal (http://cbioportal.org). Our analyses relied exclusively upon patient data which are publicly available. More specifically, reverse phase protein array (RPPA) data were obtained from 131 bladder urothelial carcinomas (Bladder Urothelial Carcinoma TCGA-Nature 2014 cohort)7, while RNA-seq data were retrieved from 407 bladder tumor samples (Bladder Urothelial Carcinoma TCGA-PanCancer Atlas cohort)57.

Drugs

PDK1 inhibitor GSK2334470 (Selleck Chemicals, Houston, TX, USA; Cat. No S7087) was used at 30 μM in cell cycle and western blot analysis. Volasertib (Selleck Chemicals; Cat. No S2235) was used at 1 μM in DMSO for synchronized culture experiments, and at 10 and 30 μM in DMSO in senescence studies using asynchronous cultures.

Cell culture

All cell lines were originally purchased from ATCC and regularly tested for mycoplasma. Cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, cat. D6429) supplemented with 10% heat-inactivated fetal bovine serum (Biosera, cat. FB-1001/500) and penicillin (100 units/ml)/streptomycin (100 μg/ml) (Thermo Fischer Scientific, cat. 15140122) at 37 °C with 5% CO2. All KMT2C knockdown (KMT2C/KD1 and KMT2C/KD2) cell lines have been described14. For live cell imaging experiments, HTB9 and T24 bladder cancer cell lines stably expressing histone H2B-mCherry were generated by lentivirus mediated transduction (pLenti6-H2B-mCherry; Addgene #89766). For the comparative analysis of γH2AX levels among different cell cycle phases, the cell cycle reporter FastFUCCI58 was used for the lentivirus transduction of HTB9 and T24 bladder cancer cell lines (Addgene #86849). For the specific analysis of γH2AX foci in S-phase, the mTagRFP-PCNA cassette from the mTagRFP-T2-PCNA-19 plasmid (Addgene #58043) was cloned to the backbone of pLenti-MP2 lentivirus plasmid (Addgene #36097). The resulted construct was used for the lentivirus transduction of HTB9 and T24 bladder cancer cell lines.

G2 checkpoint inhibition assay

Asynchronous cell cultures were irradiated with 1Gy at a dose rate of 1Gy/5 min using a GammaCell 220 irradiator (Atomic Energy of Canada Ltd., Ottawa, Canada) at room temperature. Cells were left to recover for 30 min to allow division of cells that were irradiated at mitosis and then colcemid was added to the cell cultures for 60 min. At 90 min post-irradiation, cells were then collected by centrifugation, treated in 75 mM KCl for 10 min, fixed in methanol: glacial acetic acid (3:1 v/v) and processed for preparation of metaphase chromosome spreads. The mitotic index was calculated as percent of metaphases per total nuclei number using light microscopy coupled to an image analysis system (MetaSystems, Altlussheim, Germany) to facilitate scoring.

Cell division progression

Cells constitutively expressing histone H2B-mCherry were cultured in 35 mm imaging dishes with a polymer coverslip bottom for high-end microscopy (MatTek, Ashland, MA, USA) and synchronized by thymidine treatment. To monitor the mitotic progression, prophase cells in synchronized cultures were followed until the alignment of chromosomes at the metaphase plate was complete. Cells were monitored by time-lapse video microscopy and phase fluorescence photographs were acquired every 5 min for an observation period of 300 min using a Hamamatsu Orca-ER CCD camera, connected to a Zeiss 200M inverted microscope, all controlled with the SlidebookTM 6.0 software (3i, Göttingen, Germany). The data were compacted as a video with the SlidebookTM 6.0 software and processed using the ImageJ public domain software (NIH). The duration of mitotic progression was quantitatively measured until the onset of chromosome separation in anaphase.

Real-time qPCR

For human tissue samples, total RNA was isolated with the use of TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). Reverse transcription was performed with MMLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) or PrimeScriptTM RT reagent Kit (Takara, RR037A) using oligo-dT and random primers. Quantitative PCR was performed in the 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) with Kapa SYBR Fast Universal 2× qPCR Master Mix (Kapa Biosystems, Inc., Woburn, MA, USA). Normalization was performed against the average of β-actin and GAPDH expression for Fig. 2a and against the average of β-actin and HPRT expression for Fig. 2e and S2D. Oligonucleotide sequences are provided in Table S2.

Protein extraction and western blot analysis

Cells were lysed in RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail (Complete, Roche). A total protein amount of 20 µg from each sample was denatured at 95 °C for 10 min in Laemmli buffer containing β-mercaptoethanol before electrophoresis. Antibodies used can be found in Table S3.

Immunofluorescence experiments

Cells were plated on poly-L-lysine (Sigma, cat. P1274) coated coverslips. To quantify phospho-H2AX or MRE11 foci and detect phospho-histone H3 levels, cells were fixed by 10 min incubation in 4% paraformaldehyde (Alfa Aesar, 30525-89-4) at room temperature, permeabilized for 4 min in 1× PBS/0.5% Triton X-100, washed with PBS and blocked in 1% bovine serum albumin (Applichem, cat. A1391,0100), 10% fetal bovine serum in PBS. Cells were incubated with primary antibody overnight at 4 °C, followed by incubation with a fluorescent secondary antibody for 1 h at room temperature. Antibody solutions were made in PBS with 1% bovine serum albumin. Coverslips were mounted on glass slides using VECTASHIELD Antifade Mounting Medium with 49,6-diamidino-2-phenylindole (DAPI) for DNA staining (Vektor, cat. H-1200). Antibodies used can be found in Table S3.

Senescence-associated b-galactosidase (SA-b-gal) staining

Cells were washed three times with cold PBS and then fixed with 2% formaldehyde/0.2% glutaraldehyde for 5 min at RT. After fixation, the cells were washed three times with cold PBS and then stained with freshly prepared SA--gal staining solution (40 mM citric acid/sodium phosphate buffer pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2, 1 mg/1 mL 5-bromo-4-chloro-3-indolyl- -D-galactoside) overnight at 37 °C. At least 300 cells were counted by two independent researchers. The number of SA-β-gal positive cells was expressed as a percentage of all cells counted.

GL-13 staining

GL13 staining was performed according to the manufacturer’s protocol. Formalin-fixed paraffin tumor sections were obtained from vehicle and volasertib treated xenografts mice that were generated by control and KMT2C/KD HTB9 cells. In brief, sections were deparaffinized, followed by heat-induced antigen retrieval with citrate buffer (pH 6) for 20 min using a pressure cooker. Endogenous peroxidase activity was blocked with hydrogen peroxide for 40 min followed by PBS washes and then 50% EtOH washes. Sections were incubated with SenTraGor (GL13) for 8min at RT followed by 50% EtOH washes and then by PBS washes42. Sections were permeabilized with PBS/0.3% Triton X-100 and blocked with FBS/BSA/0.3% Triton X-100. Primary anti-biotin antibody was added for overnight incubation at 4 °C in humidified chambers. Primary anti-biotin antibody was HRP-conjugated and the signal was detected with DAB (Vector Laboratories). Sections were counterstained with Mayer’s hematoxylin and mounted with DPX. GL13 compound is commercially available as SenTraGor™ from Arriani Pharmaceuticals (Cat no: AR8850040).

Cell cycle analysis

Single cell suspensions were generated with trypsinization and fixed with ethanol overnight before stained with propidium iodide (50 mg/ml, Biotium, cat. 40017) for 30 min. Flow cytometry was performed on a FC500 Beckman Coulter and for the analysis FlowJo v.10 was used.

ChIP-seq and RNA-seq experiments

All primary data and analyses have been previously described14.

Statistical analysis

In human tissue samples, the normality of the distribution of KMT2C expression in bladder tissue specimens was evaluated by Shapiro-Wilk test (p < 0.05). The correlation of KMT2C expression with tumor grade and stage was assessed by Mann-Whitney U and Kruskal-Wallis tests, respectively. One-way ANOVA was used in X-gal experiments. Kaplan-Meier survival curves, evaluated by the log-rank (Mantel-Cox) test, were used for the survival analysis of the bladder cancer patients following treatment and animals. For animal Kaplan-Meier curves, time post BBN treatment was used. Animals were randomly assigned into different groups. Group allocation and outcome assessment was not blinded. No animal was excluded. In two group comparisons, normality of distribution was determined by D’Agostino & Pearson omnibus normality test, Shapiro-Wilk normality test (paired t-test). Sample sizes met the minimum requirements of the respective statistical test used. A value of P < 0.05 was considered as significant.