Targeting MCM6 Enhances Melphalan Chemosensitivity in Retinoblastoma by Modulating DNA Damage Response

Abstract

Purpose

This study aimed to investigate the role of minichromosome maintenance complex component 6 (MCM6), a DNA replication licensing factor, in retinoblastoma progression and its impact on melphalan chemosensitivity.

Methods

MCM6 expression patterns were analyzed using single-cell RNA sequencing (scRNA-seq) of retinoblastoma and validated in patient tumors, including specimens obtained after failed melphalan therapy. Stable MCM6 knockdown cell lines were established for proliferation and cell-cycle assays, DNA damage analyses, and chemosensitivity testing. In vivo xenograft models were employed to evaluate the therapeutic efficacy of MCM6 knockdown combined with melphalan.

Results

The scRNA-seq revealed that MCM6 was highly expressed in retinoblastoma cells and embedded in a proliferation-associated gene network. Elevated expression was also confirmed in human retinoblastoma, particularly in tumors from patients with failed melphalan therapy. MCM6 knockdown suppressed cell proliferation and cell-cycle progression while enhancing melphalan-induced DNA damage, thereby sensitizing retinoblastoma cells to melphalan. In vivo, MCM6 depletion synergized with melphalan to significantly inhibit intraocular tumor growth.

Conclusions

MCM6 acts as a critical regulator of retinoblastoma growth and modulates response to melphalan. Targeting MCM6 may offer a therapeutic approach to improve outcomes of chemotherapy in retinoblastoma.

Retinoblastoma is the most common primary intraocular malignancy in children, severely threatening vision and survival.^1–5 Current treatment strategies include systemic intravenous chemotherapy (IVC), intra-arterial chemotherapy (IAC), intravitreal chemotherapy (IVitC), and enucleation.¹ Melphalan, as an DNA alkylating agent that causes deformation in the double helix of DNA,⁶ is the main chemotherapeutic agent used in both IVitC and IAC.^1,7 Although these therapeutic approaches have markedly improved eye-preservation rates, some patients still harbor residual tumor cells after chemotherapy, leading to treatment failure and eventual enucleation.^8–11 This highlights the need to identify safe and effective therapeutic targets to improve chemosensitivity in retinoblastoma.

Minichromosome maintenance complex component 6 (MCM6), a core subunit of the MCM2–7 helicase complex, plays a pivotal role in DNA replication initiation, genome stability, cell-cycle progression, and drug response across various tumors.^12–15 Previous studies have demonstrated that MCM6 knockdown induces DNA replication stress and DNA damage,^10–12 findings that closely align with the cytotoxic mechanism of melphalan. Thus, we aimed to elucidate the functional role of MCM6 in retinoblastoma and to investigate its potential contribution to the chemotherapeutic response.

In this study, we integrated single-cell transcriptomics analyses with patient tissue samples and identified increased expression of MCM6 in retinoblastoma, particularly in cases of melphalan treatment failure. In vitro and in vivo experiments further validated its role in modulating the response of retinoblastoma to melphalan, mainly through regulation of the DNA damage response and suppression of tumor cell proliferation. Together, these findings support MCM6 as a promising target to enhance melphalan chemosensitivity in retinoblastoma.

Methods

Clinical Samples

Human retinoblastoma paraffin-embedded tissue sections were obtained from the Department of Pathology, Zhongshan Ophthalmic Center, Sun Yat-sen University (Guangzhou, China). All procedures were approved by the Ethics Committee of Zhongshan Ophthalmic Center (approval no. 2023KYPJ308) and conducted in accordance with the tenets of the Declaration of Helsinki.

Single-Cell RNA Sequencing Data Processing and Integration

Single-cell RNA sequencing (scRNA-seq) data were analyzed as previously described.¹⁶ We utilized data from four retinoblastoma samples from our previous cohort8 (GEO: GSE249995),¹⁶ seven retinoblastoma samples (GEO: GSE168434),¹⁵ and five normal retina samples^17,18 (ArrayExpress: E-MTAB-7316). Gene set enrichment analysis (GSEA) was performed to compare differentially expressed genes between retina and retinoblastoma samples using the clusterProfiler 4.0 package in R (R Foundation for Statistical Computing, Vienna, Austria) with the MSigDB hallmark gene set (h.all.v7.1.symbols.gmt). High-dimensional weighted gene co-expression network analysis (hdWGCNA 0.3.03) was applied to construct a co-expression network focused on retinoblastoma (RB) cells. The module containing MCM6 was subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Escherichia coli synthetic genetic array (eSGA) enrichment analysis.

Pan-Cancer Analysis of MCM6 Using The Cancer Genome Atlas Data

To investigate the broader oncogenic relevance of MCM6, we analyzed pan-cancer expression and clinical outcome data from The Cancer Genome Atlas (TCGA). Tumor samples from 33 cancer types were stratified into MCM6-high and MCM6-low groups based on median expression. Pathway enrichment analysis was performed via single-gene GSEA using log₂ fold-change ranked gene lists. Pathways with nominal P < 0.05 were considered significant; non-significant cancer types were excluded. The top 21 enriched pathways were visualized as a –log₁₀(P) heatmap. Analyses were conducted in R using the limma, clusterProfiler, ggplot2, and pheatmap packages.

Cell Lines

Human retinoblastoma cell lines WERI-Rb1 and Y79, the retinal pigment epithelial cell line ARPE-19, and HEK293T cells were utilized in this study. WERI-Rb1 and Y79 cells were maintained in RPMI 1640 Medium (10-040-CV; Corning, Inc., Corning, NY, USA) supplemented with 10% Gibco fetal bovine serum (FBS; 10099141C; Thermo Fisher Scientific, Waltham, MA, USA) and 1% Gibco penicillin–streptomycin (15140122; Thermo Fisher Scientific). ARPE-19 cells were grown in Gibco Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium (11320033; Thermo Fisher Scientific) with 10% FBS. HEK293T cells were cultured in Gibco high-glucose DMEM (11965092; Thermo Fisher Scientific) containing 10% FBS and 1% penicillin–streptomycin. All cells were incubated at 37°C in a humidified atmosphere with 5% CO₂, and the culture medium was replaced every 2 to 3 days.

Lentiviral Infection

Lentiviruses encoding short hairpin RNA (shRNA) targeting MCM6 (shMCM6) or negative control (shNC) were packaged in HEK293T cells. By co-transfecting pLKO.1-shRNA, psPAX2, and pMD2.G (3:2:1 ratio) using polyethylenimine (PEI; 24765; Polysciences, Warrington, PA, USA). Supernatants were collected at 48 and 72 hours post-transfection, filtered through a 0.45-µm membrane, and stored at −80°C. The shRNA sequences were as follows: shMCM6-1, 5′-TGAGATGAGTCAAGATAAA-3′; shMCM6-2, 5′-GGAACAAUUUAACCAGCAATT-3′. For infection, WERI-Rb1 and Y79 cells were seeded in six-well plates and infected with viral supernatant (1:1 with fresh medium) for 24 hours. After media replacement, puromycin (1 µg/mL, HYP084; HUAYUN, Guangzhou, China) was added for 7 days to select stable lines. Knockdown efficiency was verified by western blotting.

Orthotopic Xenograft Model

Animal experiments were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center (Z2024096). BALB/c nude female mice (18–20g, 4–6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). Using a 33-gauge Hamilton syringe, 2 × 10⁵ WERI-Rb1 cells (shNC or shMCM6) in 2 µL PBS were injected intravitreally into the left eye. On day 14 post-injection, mice received a single intravitreal injection of 1-µL melphalan (0.25 µg/µL in PBS, HY-17575; MedChemExpress, Monmouth Junction, NJ, USA) or vehicle. All mice were euthanized on day 28, and eyes were enucleated, fixed in FAS Eyeball Fixative Solution (G1109; Servicebio, Wuhan, China), and paraffin-embedded for analysis.

Histology, Immunohistochemistry, and Immunofluorescence

Hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and immunofluorescence (IF) were performed according to standard protocols. Paraffin-embedded tissue sections (4 µm) were used for both H&E and IHC staining. For IHC, sections were deparaffinized, subjected to antigen retrieval (citrate buffer, pH 6.0), and blocked before incubation with primary antibodies against MCM6 (1:50, R22485; ZenBio, Durham, NC, USA) or Ki67 (1:800, GB111499; Servicebio). After washing, sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, followed by diaminobenzidine (DAB) chromogen development and hematoxylin counterstaining. For IF, cells were seeded onto poly-d-lysine (PDL)-coated glass coverslips (MedChemExpress), fixed with 4% paraformaldehyde, permeabilized, and incubated with anti-phospho-histone H2A.X (Ser139) antibody (1:1000, ET1602-2; Huabio, Woburn, MA, USA). Fluorescence images were captured using a ZEISS LSM 980 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).

Western Blotting

Cells were lysed on ice using radioimmunoprecipitation assay (RIPA) buffer (WB3100; NCM Biotech, Newport, RI, USA) with protease and phosphatase inhibitors (1:100, P002; NCM Biotech). Protein concentrations were quantified by bicinchoninic acid (BCA) assay. Equal amounts of protein were mixed with SDS-PAGE loading buffer (WB2001; NCM Biotech), heated at 95°C for 10 minutes, and then separated by SDS-PAGE before transfer onto PVDF membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 hour, then incubated with primary antibodies overnight at 4°C: GAPDH (1:10,000, 10494-1-AP; ProteinTech, Rosemont, IL, USA), cyclin D1 (1:1000, R380999; ZenBio), cyclin B1(1:1000, R23324; ZenBiox); MCM6 (1:500, R22485; ZenBio), phospho-Akt (Ser473; 1:1000, 4060; Cell Signaling Technology, Danvers, MA, USA), Akt1/2/3(1:1000, JE75-09; Huabio), phosphoinositide 3-kinase (PI3K) p85/p55 (Tyr467/Tyr199; 1:1000, 341468; ZenBio), PI3 kinase p85 alpha (1:1000, R22768; Huabio), phospho-mTOR (Ser2448; 1:1000, 81670-1-RR; Proteintech), mTOR (1:1000, HA500126; Huabio), and phospho–histone H2A.X (Ser139; 1:2000, ET1602-2; Huabio). HRP-conjugated secondary antibodies (1:10,000, SA00001-2 for rabbit and SA00001-1 for mouse; ProteinTech) were incubated for 1 hour at room temperature. Signals were detected using enhanced chemiluminescent (ECL; P10300; NCM Biotech) and imaged with a Tanon 5200 system (Tanon Science & Technology, Shanghai, China). Band intensities were quantified via ImageJ (National Institutes of Health, Bethesda, MD, USA) and normalized to GAPDH.

Cell Viability Assay

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) kit (A311-01; Vazyme, Nanjing, China). For proliferation assays, 5000 cells/well were seeded in 96-well plates, and 10-µL CCK-8 reagent was added at 0, 24, 48, and 72 hours, followed by incubation at 37°C in the dark for 3 hours. For melphalan sensitivity, 10,000 cells/well were treated for 24 hours with serially diluted melphalan (100.0 µM to 0 µM, 2:3 ratio). Absorbance was measured at 450 nm.

Cell Proliferation Assay

Cell proliferation was evaluated using the BeyoClick EdU Cell Proliferation Kit (C0071; Beyotime Biotechnology, Jiangsu, China) according to the manufacturer's instructions. In brief, 5  ×  10³ cells were seeded onto PDL-coated glass coverslips and incubated with 10-µM 5-ethynyl-2′-deoxyuridine (EdU) for 2 hours at 37°C. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were acquired using an XD-RFL fluorescence microscope (SDPTOP, Shanghai, China), and EdU incorporation was quantified as the percentage of EdU⁺ cells among total DAPI⁺ nuclei.

Cell Cycle Analysis by Flow Cytometry

Cell cycle distribution was determined using a cell cycle assay kit (E-CK-A351; Elabscience, Wuhan, China). Cells were fixed in 70% ethanol at 4°C overnight, treated with RNase A, and stained with prodidium iodide (PI) solution. DNA content was analyzed by flow cytometry (FACSCanto II flow cytometer; BD Biosciences, Franklin Lakes, NJ, USA), and cell cycle phases were quantified with FlowJo software.

Comet Assays

DNA damage was evaluated using the Comet Assay Kit (C2041S; Beyotime Biotechnology). Cells were either untreated or treated with 10-µM melphalan for 24 hours, suspended in low melting point agarose, and spread on slides. After lysis and alkaline electrophoresis (25 V, 30 minutes), slides were stained with PI solution. Images were captured via fluorescence microscope, and tail DNA percentage was quantified using Comet Assay Software Project (CASP) 1.2.3 beta2.¹⁹

Statistical Analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using Prism 10 (GraphPad, Boston, MA, USA). Unpaired two-tailed Student's t-tests were used for two-group comparisons, and one-way or two-way ANOVA with Tukey's post hoc test was used for multiple groups. P < 0.05 was considered statistically significant. Each experiment included at least three biological replicates.

Results

Single-Cell RNA-Seq Reveals High MCM6 Expression Across Retinoblastoma Cell Populations

To characterize the expression pattern of MCM6 in retinoblastoma at single-cell resolution, we analyzed 16 scRNA-seq datasets, including five normal human retina samples and 11 human retinoblastoma samples. Dimensionality reduction and clustering (t-distributed stochastic neighbor embedding [t-SNE]/Uniform Manifold Approximation and Projection [UMAP]) revealed distinct transcriptional profiles between normal and tumor tissues, with nine annotated cell clusters, including cone and rod precursor-like cells, rods/rod-like cells, cones/cone-like cells, Müller glia, microglia, bipolar cells, retinoma-like cells, and MKI67⁺ photoreceptorness decreased (PhrD) cells (Figs. 1A, 1B). Differential expression analysis revealed that MCM6 was one of the most significantly upregulated genes in retinoblastoma compared to normal retina (Fig. 1C). Violin plots further showed markedly higher MCM6 expression in both intraocular and extraocular retinoblastoma compared to normal retina (Fig. 1D). Single-cell visualization confirmed minimal MCM6 expression in normal retinal cells but broad, high-level expression across retinoblastoma cells (Fig. 1E). Consistently, dot plot analysis further demonstrated preferential enrichment of MCM6 in different cell clusters within retinoblastoma compared to their retinal counterparts (Fig. 1F). Furthermore, IHC analysis revealed strong nuclear MCM6 staining in retinoblastoma tissues, in contrast to weak staining in normal retina (P < 0.0001) (Fig. 1G). Western blot further showed robust MCM6 expression in WERI-Rb1 and Y79 cells, but low expression in ARPE-19 (Fig. 1H). Collectively, these results demonstrate that MCM6 is broadly upregulated in retinoblastoma.

Figure 1.

Expression pattern of MCM6 in retinoblastoma. (A) UMAP plots showing the distribution of cell populations in normal retina and retinoblastoma tissues. (B) UMAP plots of subtype cell clusters in retinoblastoma, with different colors representing various cell types. (C) Volcano plot showing differentially expressed genes between normal retina and retinoblastoma tissues, with MCM6 specifically highlighted. (D) Violin plot of MCM6 expression levels in Normal, Intraocular, and Extraocular retinoblastoma groups. (E) UMAP plots showing the single-cell distribution of MCM6 in normal retina and retinoblastoma samples. (F) Bubble plot illustrating MCM6 expression across retinoblastoma cellular subtypes and grouped by normal, intraocular, and extraocular retinoblastoma. (G) Representative immunohistochemical staining images of MCM6 in human retina and retinoblastoma tissues, along with quantification of MCM6 IHC staining (integrated optical density/area). Data are presented as mean ± SD (n = 8), with significance assessed by unpaired two-tailed Student's t-test. ****P < 0.0001. (H) Western blot analysis of MCM6 protein levels in ARPE-19, WERI-Rb1, and Y79 cell lines and grayscale analysis. Data are presented as mean ± SD (n = 3), with significance assessed by ordinary one-way ANOVA. **P < 0.01. DEGs, differentially expressed genes; MKI67⁺PhrD, MKI67⁺ photoreceptorness decreased.

MCM6 Persists After Melphalan Chemotherapy

To further validate the expression of MCM6 in chemoresistant residual retinoblastoma cells after failed melphalan treatment, we performed IHC on tumor tissues from six patients who did not respond to melphalan IVitC. The results demonstrated that MCM6 remained strongly expressed in the residual tumor cells (Fig. 2A), and the percentages of MCM6⁺ cells were significantly higher than in untreated RB cases (Fig. 2B). The corresponding clinicopathological features and treatment details are summarized in the Table.

Figure 2.

Expression of MCM6 after intravitreal melphalan chemotherapy in human retinoblastoma. (A, B) Immunohistochemical staining of MCM6 in human RB tissues from melphalan (Melp)-refractory cases (A) and percentage of MCM6⁺ cells compared with untreated RB tissues (B). Data are presented as mean ± SD (n = 8 for untreated group; n = 6 for melphalan-refractory group), with significance assessed by unpaired two-tailed Student's t-test. **P < 0.01.

Table.

Clinicopathological Features and Treatments of Retinoblastoma Patients

			IVitC Dose (µg)/Injections, n
Patient No.	IIRC	AJCC	Melphalan	Topotecan	CEV Regimen (Cycles), n
Group 1: Patients 1–8 (primary enucleation without chemotherapy)
1	E	T2a	NA	NA	NA
2	E	T2b	NA	NA	NA
3	D	T2a	NA	NA	NA
4	E	T4a	NA	NA	NA
5	E	T3c	NA	NA	NA
6	D	T2b	NA	NA	NA
7	E	T2b	NA	NA	NA
8	E	T4a	NA	NA	NA
Group 2: Patients 9–15 (enucleation after failure of intravitreal melphalan)
9	E	T2b	30/4	NA	5
10	E	T4a	30/3	50/1	3
11	E	T3a	30/2	NA	2
12	E	T3d	40/2	NA	1
13	D	T2a	30/3; 40/1	NA	3
15	E	T3b	30/8	NA	5

AJCC, American Joint Committee on Cancer classification; CEV, carboplatin, etoposide, and vincristine intravenous chemotherapy; IIRC, International Intraocular Retinoblastoma Classification; IVitC, intravitreal chemotherapy; NA, not applicable.

MCM6 Knockdown Enhanced Retinoblastoma Sensitivity to Melphalan

To determine whether MCM6 knockdown could potentiate the cytotoxic effect of melphalan, we generated stable MCM6 knockdown (shMCM6-1 and shMCM6-2) cell lines. Western blot analysis confirmed efficient knockdown of MCM6 in both WERI-Rb1 and Y79 cell lines compared to the negative control (Figs. 3A, 3B). MCM6 knockdown markedly enhanced melphalan sensitivity, reducing the 24-hour half-maximal inhibitory concentration (IC₅₀) of melphalan from 21.34 µM to 8.46 µM in WERI-Rb1 cells and from 19.76 µM to 5.92 µM in Y79 cells (Fig. 3C). In vivo, BALB/c nude mice were intravitreally injected with WERI-Rb1 shNC or shMCM6-1 cells, followed by intravitreal administration of melphalan or PBS. On day 28, eyes were enucleated for pathological analysis. H&E staining showed that both melphalan alone and MCM6 knockdown alone significantly reduced intraocular tumor burden, although small numbers of residual tumor cells remained. Remarkably, the combination of MCM6 knockdown and melphalan led to a dramatic reduction of residual tumor cells, with almost no detectable tumor remaining (Figs. 3E, 3F). Consistently, Ki67 immunostaining revealed high proliferative activity in the negative control group and substantial Ki67 expression in the residual cells of the melphalan-only group. In contrast, Ki67 staining was reduced in the MCM6 knockdown group and completely absent in the combination group, indicating effective elimination of proliferating tumor cells (Figs. 3G, 3H). Collectively, these findings demonstrate that MCM6 knockdown synergistically enhances the therapeutic efficacy of melphalan both in vitro and in vivo.

Figure 3.

MCM6 knockdown enhances sensitivity of RB cells to melphalan. (A, B) Western blot analysis validating the knockdown efficiency of MCM6 in WERI-Rb1 cells. (C) Cell viability assays and IC₅₀ analysis of melphalan treatment in WERI-Rb1 and Y79 cells with or without MCM6 knockdown. Data are presented as mean ± SD (n = 3), with IC₅₀ values derived from nonlinear regression (four-parameter logistic model). (D) Schematic diagram of the experimental design. (E, F) Gross and H&E staining images of enucleated eyes from different experimental groups (E) and quantification of tumor area in NC and shMCM6 groups without and with melphalan treatment (F). Data are presented as mean ± SD (n = 5), with significance assessed by unpaired two-tailed Student's t-test. **P < 0.01, ***P < 0.001. (G, H) Immunohistochemical staining of Ki67 in NC and shMCM6 groups without and with melphalan treatment (G) and quantification of Ki67 levels (H). Data are presented as mean ± SD (n = 3), with significance assessed by unpaired two-tailed Student's t-test. **P < 0.01.

MCM6 Knockdown Disrupts Cell Cycle Progression in Retinoblastoma

We further investigated the functional role of MCM6 in retinoblastoma cells. EdU incorporation assays revealed a marked decrease in the proportion of EdU⁺ proliferating cells following MCM6 depletion (Figs. 4A, 4B). Consistently, CCK-8 assays showed that MCM6 knockdown significantly reduced cell proliferation in both retinoblastoma cell lines (Fig. 4C). Flow cytometry analysis demonstrated that MCM6 silencing decreased the S-phase population while inducing G2-phase accumulation in WERI-Rb1 and Y79 cells (Figs. 4D–G). In vivo, IHC analysis showed that MCM6 knockdown impaired local invasion. The shMCM6 tumors exhibited limited invasion compared to shNC tumors, which frequently invaded the sclera and cornea and extended beyond the globe (Figs. 4H–K). These results demonstrate that MCM6 is critical for retinoblastoma cell proliferation, cell cycle progression, and invasiveness.

Figure 4.

MCM6 knockdown disrupts cell cycle progression and inhibits in vivo invasion. (A, B) EdU incorporation assay showing the proliferation of WERI-Rb1 cells (A) and Y79 cells (B) following MCM6 knockdown. (C) WERI-Rb1 and Y79 cell proliferation assessed by CCK-8 assay after MCM6 knockdown. Data are presented as mean ± SD (n = 3), with significance assessed by two-way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001. (D–G) Flow cytometry analysis of cell cycle distribution in WERI-Rb1 cells (D) and Y79 cells (F) after MCM6 knockdown and quantification of the proportion of cells in G1, S, and G2 phases, respectively. In E and G, data are presented as mean ± SD (n = 3), with significance assessed by ordinary one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant. (H) H&E observation of tumor invasion: H&E-stained sections depict scleral invasion, cornea invasion, and extraocular stage in NC and shMCM6 groups. (I–K) Statistical analysis of tumor invasion: Proportion of eyes with anterior chamber (Ac) involvement (I), eyeball wall invasion (J), and extraocular stage (K) in negative control and shMCM6 groups.

MCM6 Integrates Into a DNA Replication–Cell Cycle Gene Network in Retinoblastoma

To uncover the molecular mechanisms by which MCM6 drives retinoblastoma malignant progression, we performed multi-omics analyses. First, pan-cancer GSEA across TCGA data revealed that MCM6 expression strongly correlates with cell cycle and proliferation pathways, including MITOTIC_SPINDLE, E2F_TARGETS, G2M_CHECKPOINT, MYC_TARGETS_V1/V2, and MTORC1_SIGNALING, among the top pathways (Fig. 5A). Interestingly, focusing on retinoblastoma, GSEA using scRNA-seq data (normal retina vs. retinoblastoma) confirmed that retinoblastoma samples were significantly enriched in the same cell cycle–associated gene sets (Fig. 5B). This overlap suggests that the proliferative programs commonly associated with MCM6 in diverse cancers are also dominant transcriptional features of retinoblastoma, providing a biological context for its potential role in retinoblastoma progression. To further elucidate the functional network associated with MCM6 in retinoblastoma, we performed hdWGCNA on three major retinoblastoma subtype cell clusters, including cones/cone-like cells, MKI67⁺ PhrD cells, and retinoma-like cells. The resulting dendrogram delineated multiple co-expression modules (Fig. 5C), and UMAP mapping localized MCM6 within a “orange–red module” (Fig. 5D). As expected, GSEA enrichment showed that MCM6-associated genes were significantly enriched in the E2F_TARGETS, MYC_TARGETS_V1, and DNA_REPAIR pathways (Fig. 5E). Moreover, KEGG and GO enrichment of genes in this module confirmed its identity as a cell proliferation–related module (Figs. 5F, 5G).

Figure 5.

MCM6 knockdown inhibits cell cycle progression and inactivates the PI3K/Akt/mTOR signaling pathway. (A) Heatmap showing GSEA enrichment scores for MCM6 expression across multiple cancer types from the TCGA database. (B) GSEA results were obtained from differentially expressed genes between normal retina and RB using scRNA-seq data. (C) Dendrogram of hdWGCNA in RB cells. (D) UMAP plot displaying the single-cell co-expression distribution of MCM6 in retinoblastoma. (E) GSEA enrichment plots showing hallmark gene sets significantly correlated with the MCM6 co-expression module, including E2F_TARGETS, MYC_TARGETS_V1, and DNA_REPAIR repair pathways. (F) KEGG pathway enrichment of genes co-expressed with MCM6. (G) GO functional enrichment of genes positively correlated with MCM6. (H–K) Western blot analysis of cyclin D1, cyclin B1, and PI3K/Akt/mTOR signaling proteins after MCM6 knockdown in WERI-Rb1 and Y79 cells. Data are presented as mean ± SD (n = 3), with significance assessed by unpaired two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001. NES, normalized enrichment score; BP, biological process; MF, molecular function; CC, cellular component.

To validate these findings experimentally, we confirmed these bioinformatic insights via MCM6 knockdown in WERI Rb1 and Y79 cells. Western blot showed that MCM6 knockdown significantly reduced expression of core cell cycle regulators cyclin D1 and cyclin B1 (Figs. 5G, 5H), consistent with impaired G1/S and G2/M transitions. Moreover, signaling analyses revealed that depletion of MCM6 markedly decreased the phosphorylated forms of PI3K, Akt, and mTOR (Figs. 5I, 5J), indicating that MCM6 is required to sustain PI3K/Akt/mTOR pathway activity in retinoblastoma cells. Overall, these results position MCM6 as a central regulator of proliferation in retinoblastoma and maintaining PI3K/Akt/mTOR signaling.

MCM6 Knockdown Potentiates Melphalan Efficacy by Suppressing Survival Signaling and Exacerbating DNA Damage

In order to elucidate how MCM6 modulates retinoblastoma cell sensitivity to melphalan, we assessed its effects on PI3K/Akt survival signaling and the DNA damage response. In both retinoblastoma cell lines, 10-µM melphalan for 24 hours reduced PI3K and Akt phosphorylation, and MCM6 knockdown further reinforced this suppression (Figs. 4A, 4B). In parallel, the DNA damage marker γ-H2A.X was markedly upregulated in the shMCM6⁺ melphalan group, as confirmed by both western blot and IF staining of γ-H2A.X foci (Figs. 4A–C). Quantification revealed a modest increase with MCM6 knockdown alone (significant in Y79, trending in WERI-Rb1), but the combination with melphalan induced a strong and highly significant increase in both cell lines (P < 0.0001) (Fig. 6D). Comet assays further corroborated these findings, showing that the tail DNA percentage was highest in the shMCM⁺ melphalan group, with knockdown alone also producing a measurable increase (Figs. 4E, 4F). Together, these data established that MCM6 knockdown markedly augments the cytotoxicity of melphalan by concurrently disabling PI3K/Akt survival signaling and intensifying DNA damage. These findings indicate that MCM6 knockdown sensitizes retinoblastoma cells to melphalan by concurrently disabling survival signaling and enhancing DNA damage.

Figure 6.

MCM6 knockdown enhances melphalan sensitivity by promoting DNA damage and inhibiting the PI3K/Akt pathway. (A, B) Western blot analysis of phosphorylated and total mTOR, PI3K, and Akt, as well as γ-H2A.X expression, in WERI-Rb1 cells (A) and Y79 cells (B) following MCM6 knockdown and/or melphalan treatment. (C, D) IF staining (C) and quantification (D) of γ-H2A.X foci in WERI-Rb1 and Y79 cells under the indicated treatments. (E, F) Comet assay images (E) and quantification of tail DNA percentage (F) in WERI-Rb1 and Y79 cells. In D and F, data are presented as mean ± SD (n = 3), with significance assessed by ordinary one-way ANOVA. *P < 0.05, ***P < 0.001, ****P < 0.0001; NS, not significant.

Discussion

In this study, we identified MCM6 as a potent modulator of chemotherapeutic response. By integrating single-cell transcriptomics, we found that MCM6 is markedly upregulated in retinoblastoma compared with normal retina and integrated into proliferation-associated transcriptional networks. Functionally, MCM6 sustains tumor growth by maintaining cell cycle progression and supporting the PI3K/Akt/mTOR pathway. Importantly, MCM6 knockdown markedly enhanced the efficacy of melphalan by potentiating chemotherapy-induced DNA damage while concomitantly attenuating PI3K/Akt activation. These findings position MCM6 as a promising therapeutic target, capable of both inhibiting tumor growth and enhancing chemotherapeutic efficacy in retinoblastoma.

Melphalan is one of the most widely used anticancer agents, as it has broad activity against various malignancies including multiple myeloma, ovarian cancer, breast cancer, and retinoblastoma.^1,6,7 As a nitrogen mustard alkylating agent, it exerts cytotoxic effects primarily through DNA alkylation and interstrand crosslinking, leading to replication and transcription blockade, aberrant DNA repair, accumulation of DNA strand breaks, and ultimately cell death.⁶ Although melphalan has achieved remarkable success in the treatment of retinoblastoma, not all patients respond favorably to this drug. A subset still experiences treatment failure and ultimately requires enucleation. Additionally, there are ongoing concerns about melphalan IVitC side effects, as retinal toxicity affects roughly 36.56% of patients, which is particularly notable in Asian populations.^1,20–22 Thus, reducing melphalan dosage while improving its antitumor efficacy has become a pressing clinical objective.

Building on these clinical challenges, our clinical observations showed persistent high MCM6 expression in residual vitreous seeds after intravitreal chemotherapy, suggesting that MCM6 could be a potential mediator of chemoresistance (Fig. 2). To determine whether its inhibition could enhance chemosensitivity in retinoblastoma, we evaluated the effects of MCM6 knockdown in a xenograft mouse model (Fig. 3). The combination of MCM6 knockdown and melphalan resulted in the most pronounced tumor regression, markedly outperforming either monotherapy. This synergistic effect was evidenced by the greatest reduction in tumor burden and lowest expression of the proliferation marker Ki67 across all groups. In vitro, knockdown of MCM6 reduced the IC₅₀ of melphalan by over 50% in both WERI-Rb1 and Y79 cell lines (Fig. 3C). These findings underscore the clinical relevance of targeting MCM6 by demonstrating that its inhibition potently enhances the antitumor activity of melphalan, highlighting a synergistic strategy to achieve deeper tumor regression in retinoblastoma.

Considering these findings, it is important to contextualize MCM6 within the molecular framework of retinoblastoma tumorigenesis. Biallelic RB1 inactivation, the earliest and most common driver, causes retinoblastoma protein (pRB) loss, disrupts the pRB/E2F checkpoint, and leads to constitutive activation of E2F-regulated genes that drive G₁/S transition and DNA replication.^8–10 Although MYCN amplification occurs in only 1% to 9% of retinoblastoma cases, these tumors display early-onset, aggressive growth driven by MYCN-mediated upregulation of E2F and mitotic genes, which override RB1 control and fuel unchecked cell cycle progression.^8,9,23 Within this oncogenic framework, our scRNA-seq analysis showed significant enrichment of hallmark proliferation pathways across the retinoblastoma cell population, including MYC_TARGETS, G2M_CHECKPOINT, E2F_TARGETS, and MITOTIC_SPINDLE (Fig. 5B). This pervasive proliferative state in retinoblastoma, combined with the role of MCM6 as a core component of the replicative helicase essential for DNA replication and cell cycle progression, positions MCM6 as a critical executor of these dysregulated proliferation programs in retinoblastoma tumorigenesis.

Functionally, our results demonstrate that knockdown of MCM6 markedly impaired cell proliferation and disrupted cell cycle progression in retinoblastoma cell lines (Fig. 4). In support of these findings, pan-cancer GSEA analysis revealed a strong positive association between MCM6 expression and hallmark proliferative programs, including MITOTIC_SPINDLE, MYC_TARGETS, G2M_CHECKPOINT, E2F_TARGETS, and MTORC1_SIGNALING (Fig. 5A). These same pathways were significantly enriched in our retinoblastoma scRNA-seq dataset relative to normal retina, indicating a shared transcriptional signature of MCM6-associated proliferation across cancer types and within retinoblastoma specifically. To delineate the molecular context of MCM6 activity in retinoblastoma, hdWGCNA identified MCM6 as a node within a proliferation-enriched module (Fig. 5D). GSEA revealed that the MCM6-related module was significantly enriched in the E2F_TARGETS, MYC_TARGETS_V1, and DNA_REPAIR pathways, underscoring the strong potential of MCM6 inhibition to suppress RB cell proliferation. Moreover, KEGG pathway analysis of this module highlighted its enrichment in cell cycle and DNA metabolism–related processes. GO enrichment further demonstrated an overrepresentation of DNA replication and DNA repair pathways. These findings were further validated by MCM6 knockdown in RB cells, which resulted in the downregulation of key cell cycle regulators—cyclin D1 (upstream of E2F) and cyclin B1 (downstream of E2F).^24–26 Collectively, these multilevel enrichment results highlight the likely involvement of MCM6 in orchestrating genome replication, stability maintenance, and cell cycle progression—core processes driving dysregulated proliferation in retinoblastoma.

Importantly, our study revealed that MCM6 knockdown markedly amplified melphalan-induced DNA damage in retinoblastoma cells, as evidenced by increased γ-H2A.X accumulation and comet tail formation. Given the canonical role of MCM6 in DNA replication and mitotic progression, its depletion likely compromises replication integrity, thereby sensitizing cells to chemotherapy-induced DNA damage. This chemosensitization likely stems from impaired replication licensing upon MCM6 loss, which eliminates the reserve of dormant replication origins normally recruited for replication fork recovery and DNA repair under genotoxic stress, thus dismantling a key resistance mechanism to melphalan.^14,27,28 We also observed reductions in PI3K, Akt, and mTOR phosphorylation, indicative of suppression of a key pro-survival signaling axis.^29–32 Previous studies have implicated MCM6 in diverse oncogenic functions beyond replication control, such as promoting p53 degradation and activating PI3K/AkT signaling via yes-associated protein (YAP) regulation, underscoring its versatility as a cancer driver.^33,34 Our findings extend these observations to retinoblastoma. Notably, inhibition of the PI3K/Akt pathway was evident even before overt DNA damage became detectable following MCM6 knockdown. This suggests that, in retinoblastoma, MCM6 may directly modulate the PI3K/Akt signaling cascade through a mechanism independent of its canonical roles in DNA replication and damage response. Collectively, our study positioned MCM6 as a molecular hub whose inhibition simultaneously disrupts survival signaling and exacerbates DNA damage. Moreover, MCM6 is selectively overexpressed in retinoblastoma cells but minimally expressed in normal intraocular tissues, highlighting its promise as a tumor-specific therapeutic target to enhance melphalan efficacy, potentially enabling lower dosing without sacrificing potency.

Beyond MCM6, aberrant expression of other MCM family members is a common feature across diverse malignancies, driving uncontrolled proliferation and promoting genomic instability.³⁵ For example, in neuroblastoma, amplified MYCN binds to the proximal promoters of MCM2 to MCM8, thereby inducing their transcription.³⁶ In esophageal squamous cell carcinoma, MCM2 and MCM6 directly interact with DNA damage checkpoint 1, influencing the formation of damaged foci upon bleomycin-induced DNA injury.²⁷ Moreover, elevated plasma cell-free MCM2 RNA levels in metastatic neuroblastoma suggest that circulating MCM transcripts may serve as minimally invasive biomarkers,³⁷ raising the possibility that MCM6 could similarly function as a noninvasive marker in retinoblastoma. Despite their well-established oncogenic relevance, no small-molecule inhibitors directly targeting MCM proteins have yet reached clinical application. Future work should therefore focus on developing selective and potent MCM inhibitors, which could offer a promising therapeutic avenue for targeting proliferation-driven and chemoresistant tumors such as retinoblastoma.

Although we demonstrated the role of MCM6 in tumor proliferation, chemoresistance, and therapeutic potential, this study has several limitations. First, translating these findings to clinical practice faces notable hurdles. The development of MCM6-targeted agents would require rigorous preclinical evaluation of their specificity, toxicity profiles, and optimal delivery methods—particularly critical in pediatric retinoblastoma, where preserving ocular function and minimizing systemic side effects are paramount. Additionally, our current work does not fully dissect the upstream regulators and its precise interactions with other DNA replication or survival pathways remain incompletely characterized. Elucidating these regulatory networks could refine our understanding of the oncogenic role of MCM6 and identify additional co-targets to enhance therapeutic efficacy, which will be critical for advancing this research toward clinical application.

Collectively, our results identify MCM6 as a central regulator of proliferation and a key modulator of melphalan sensitivity in retinoblastoma. Through its roles in promoting oncogenic signaling and attenuating DNA damage-induced cytotoxicity, MCM6 emerges as a compelling therapeutic target.