Targeting MYCN Degradation by HLB-0532259 Induces Senescence in Retinoblastoma

Abstract

Purpose

Amplification of MYCN is a primary driver of aggressive retinoblastoma (RB), yet it remains a challenging therapeutic target. This study aimed to evaluate the therapeutic potential of the PROTAC molecule HLB-0532259 in degrading MYCN and suppressing RB growth.

Methods

Y79 and WERI-Rb1 RB cell lines were treated with HLB-0532259, and MYCN protein degradation, cell viability, and time-dependent transcriptional changes were assessed by Western blotting, cell viability assays, RNA-seq, and proteomics. The therapeutic potential was further validated in an orthotopic Y79 xenograft mouse model through intravitreal administration, with tumor burden evaluated by histology and immunohistochemistry.

Results

HLB-0532259 induced dose-dependent degradation of MYCN in both cell lines, with Y79 cells showing higher sensitivity. Transcriptomic and proteomic analyses revealed rapid activation of the p53–p21 pathway, downregulation of DNA repair and epigenetic regulators, and accumulation of DNA damage. Proteomic profiling confirmed upregulation of senescence-associated secretory phenotype factors and downregulation of MYCN-dependent effectors, including HMGA1 and TRAF6. In orthotopic xenografts, intravitreal administration of HLB-0532259 reduced intraocular tumor burden, decreased Ki-67 and MYCN-positive cells, and activated p53, p21, and NF-κB signaling.

Conclusions

Targeted MYCN degradation by HLB-0532259 effectively inhibits RB growth and induces a DNA damage–associated senescence program, highlighting targeted MYCN degradation as a promising therapeutic strategy for MYCN-driven RB.

Retinoblastoma (RB) is the most common intraocular malignancy of childhood and is primarily initiated by biallelic inactivation of the RB1 tumor-suppressor gene. Beyond RB1 loss, additional genetic alterations have been identified, including MYCN amplification (MYCN^A) and single-nucleotide variants that contribute to tumor heterogeneity.^1,2 Among these events, MYCN^A has gained increasing attention over recent decades because of its strong oncogenic potential. MYCN belongs to the MYC transcription factor family and encodes the N-Myc protein, which plays an important role in driving oncogenesis. Aberrant MYCN expression is implicated in several pediatric cancers, including RB, neuroblastoma, and medulloblastoma, as well as a range of adult malignancies.^3–5 In RB, MYCN^A represents a key genetic driver that promotes tumor progression through diverse molecular mechanisms and signaling pathways.^6–9

Accumulating evidence indicates that MYCN overexpression induces replication stress and genomic instability.¹⁰ MYCN-driven tumors rely heavily on DNA damage response mechanisms to tolerate elevated levels of replication-associated DNA damage. Experimental disruption of replication stress–mitigating pathways in MYCN^A models results in accumulation of double-strand breaks, activation of checkpoint signaling, and p53-dependent growth arrest.^11,12 These findings suggest that MYCN^A cells exist in a state of heightened proliferative and genomic stress. Persistent replication-associated damage is a recognized trigger of p53-mediated cellular senescence, indicating that perturbation of MYCN signaling may shift tumor cells from uncontrolled proliferation toward irreversible growth arrest.

Despite its central oncogenic role, MYCN has long been considered “undruggable” because of its intrinsically disordered structure and lack of well-defined ligand-binding pockets.¹³ Nevertheless, emerging evidence suggests that inducing MYCN degradation may offer a promising therapeutic strategy.¹⁴ N-Myc stability is regulated in part by its interaction with Aurora kinase A (AURKA), which protects N-Myc from ubiquitin-mediated degradation.^15,16 Although certain Aurora-A inhibitors can disrupt this interaction and promote MYCN degradation, their efficacy remains limited, particularly in tumors with high MYCN expression.¹⁷ Proteolysis-targeting chimeras (PROTACs) provide an alternative strategy by recruiting an E3 ubiquitin ligase to induce targeted proteasomal degradation of specific proteins. Recently, the PROTAC HLB-0532259 was developed using a modified AURKA-binding ligand to promote degradation of the AURKA–MYCN complex, offering a potential strategy to eliminate MYCN-driven oncogenic signaling.¹⁸

In the present study, we investigated the therapeutic effects of HLB-0532259 in RB models and examined the molecular consequences of MYCN degradation. Temporal transcriptomic analysis unexpectedly revealed coordinated activation of cellular senescence–associated pathways after MYCN loss. Mechanistic studies demonstrated rapid engagement of the p53–p21 axis, accumulation of DNA damage, and activation of senescence-related signaling both in vitro and in an orthotopic xenograft model. Together, these findings establish MYCN degradation as a trigger of p53–p21–mediated senescence in RB and provide mechanistic insight into the therapeutic potential of HLB-0532259 in MYCN-driven tumors.

Methods

Cell Lines and Reagent

WERI-Rb1 (HTB-169, American Type Culture Collection [ATCC]) and Y79 (HTB-18, ATCC) cell lines were used in this study. Cells were maintained in RPMI-1640 medium (Corning, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere containing 5% CO₂. HLB-0532259 (HY-168439; MedChemExpress, Monmouth Junction, NJ, USA) and ATM inhibitor KU-55933 (S1092; Selleck Chemicals, Houston, TX, USA) was dissolved in DMSO to a stock concentration of 1 mM and aliquoted for storage.

Cell Viability Assay

Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; A311-01; Vazyme Biotech Co., Ltd, Nanjing, China). Y79 and WERI-Rb1 cells were seeded into 96-well plates at 5000 cells/well. For IC₅₀ determination, 10 µL CCK-8 reagent was added 48 hours after seeding, followed by a four-hour incubation. Absorbance was measured at 450 nm. All experiments were performed in triplicate.

Western Blotting

Proteins from cells and tissues were extracted using RIPA lysis buffer (WB3100; NCM Biotech, Suzhou, China) supplemented with ProtLytic Protease and Phosphatase Inhibitor Cocktail (P002; NCM Biotech, Newport, RI, USA). Proteins were separated on 10% or 15% precast gels (FuturePAGE; ACE Biotechnology, Changzhou, China) and transferred to 0.2 µm PVDF membranes (Thermo Fisher Scientific). Membranes were incubated with primary antibodies against AURKA (1:5000, 82906-2-RR; Proteintech, Rosemont, IL, USA), MYCN (1:1000, 10159-2-AP; ProteinTech), SUZ12 (1:1000, 3737T; Cell Signaling Technology, Danvers, MA, USA), Phospho-Histone H2A.X(Ser139) (1:1000, 9718T; Cell Signaling Technology), NF-κB p65 (1:1000, 8242T; Cell Signaling Technology), p53 (1:1000, 2527T; Cell Signaling Technology), IκBα (1:1000, 4814T; Cell Signaling Technology), p21 (1:1000, 2947T; Cell Signaling Technology), TRAF6 (1:1000, 8028T; Cell Signaling Technology), HMGA1 (1:1000, 12094T; Cell Signaling Technology), GDF15 (1:1000, 79996T; Cell Signaling Technology), IGFBP3 (1:1000, 64143T; Cell Signaling Technology), SERPINA1 (1:1000, 55573T; Cell Signaling Technology) and GAPDH (1:10000, 10494-1-AP; ProteinTech). After incubation with HRP-conjugated secondary antibodies (1:10,000; ProteinTech), membranes were washed three times for 10 minutes with TBS-0.1% Tween-20 and visualized using enhanced chemiluminescence on the Tanon 5200 MultiImage System (Tanon, Shanghai, China).

RNA Sequencing and Analysis

Total RNA was isolated from Y79 cells treated with HLB-0532259 for 12, 24, or 48 hours, and untreated cells (0 hours). RNA sequencing was performed by Novogene (Beijing, China). RNA quality was confirmed using the Agilent 2100 Bioanalyzer (RNA 6000 Nano Kit; Agilent Technology, Tokyo, Japan). Libraries were sequenced using a 2 × 150 bp reads. Differential expression analysis was performed using DESeq2 (v1.39.8), with DEGs ranked by P-value. Time-dependent expression patterns were clustered using the Mfuzz R package to identify gene clusters with similar temporal trajectories. Functional annotation and enrichment analyses for each gene cluster were performed using the Database for Annotation, Visualization and Integrated Discovery.

Proteomics Analysis

Proteins extracted from Y79 cells treated with HLB-0532259 for 48 hours or untreated controls (0 hours) were processed and digested by Novogene. Peptides were analyzed on an Orbitrap Astral mass spectrometer (Thermo Fisher Scientific) coupled to a Vanquish Neo LC system in data-independent acquisition (DIA) mode. DIA data were processed using DIA-NN (v1.8.1). Normalized protein intensities were subjected to multivariate analysis in SIMCA-P (v14.1), including Pareto-scaled principal component analysis (PCA). Hierarchical clustering was performed using Cluster 3.0 and visualized with Java TreeView. Differentially expressed proteins were defined using a fold change (FC) > 1.5 for upregulated proteins or < 0.67 for downregulated proteins, together with a P value < 0.05 (Student's t-test). Enrichment analysis was conducted using Fisher's exact test with Benjamini–Hochberg correction, using all quantified proteins as the background set.

EdU Proliferation Assay

Cell proliferation was assessed using the BeyoClick EdU Kit with Alexa Fluor 594 (C0078; Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer's instructions. Cells were seeded onto poly-D-lysine–coated coverslips in six-well plates at a density of 1 × 10⁵ cells per well and treated with vehicle or HLB-0532259 for 24 hours. Cells were then incubated with 10 µM EdU for two hours at 37°C. After incubation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and subjected to the click reaction cocktail (100 µL per well) for 30 minutes at room temperature. Nuclei were counterstained with Hoechst 33342 (Sanofi-Aventis, Paris, France). Fluorescence images were acquired under identical exposure settings. EdU-positive nuclei (Alexa Fluor 594; Thermo Fisher Scientific) were quantified using ImageJ and expressed as a percentage of total nuclei (Hoechst-stained) from at least three independent samples.

Soft Agar Assay

Low-melting-point agarose (Agarose, Low Melting Point; ST105; Beyotime Institute of Biotechnology) was prepared at 1.2% (bottom layer) and 0.7% (top layer) using sterile distilled water. The agarose solutions were maintained at approximately 42°C to prevent solidification. For the bottom layer, equal volumes of 1.2% agarose and 2 × complete culture medium were mixed thoroughly and added to six-well plates, followed by solidification at room temperature. For the upper layer, cells were pretreated with vehicle or HLB-0532259, subjected to drug washout, and then re-seeded as single-cell suspensions at a density of 2000 cells per well. Cells were mixed with an equal volume of 0.7% agarose prepared in 2 × complete medium and overlaid onto the pre-solidified bottom layer. After solidification, 1 mL of complete 1 × culture medium was gently added on top of each well. Cells were cultured for three weeks to allow colony formation. At the end of the incubation period, colonies were stained with nitroblue tetrazolium chloride and incubated until visible colonies were developed.

Immunofluorescence and Immunohistochemistry

For immunofluorescence, Y79 cells were seeded on poly-D-lysine–coated coverslips (1 × 10⁵ cells/well) for 24 hours and then treated with HLB-0532259 for 48 hours before fixation and permeabilization. For immunohistochemistry, paraffin-embedded ocular sections were deparaffinized, rehydrated, and subjected to citrate buffer antigen retrieval (pH 6.0) followed by blocking with 5% BSA. Sections were incubated overnight at 4°C with primary antibodies against phospho-H2A.X (Ser139; 1:240), NF-κB p65 (1:400), p53 (1:200), IκBα (1:100), and p21 (1:100). Images were acquired using a Leica fluorescence microscope (Leica, Wetzlar, Germany). Positive cells were quantified in randomly selected high-power fields per section.

Animal Studies

Female BALB/c nude mice (four to six weeks old; 18–20g; Zhuhai BesTest Bio-Tech, Zhuhai, China) were maintained under specific pathogen-free conditions. Orthotopic retinoblastoma xenografts were established as previously described. Briefly, Y79 cells (1 × 10⁵ in 1 µL PBS) were injected into the vitreous cavity of the right eye using a 33-gauge Hamilton syringe; the contralateral eye served as an untreated control (n = 5 per group). To evaluate therapeutic effects, intravitreal injections of HLB-0532259 (1 µM or 10 µM) or vehicle (DMSO) were administered 14 days after tumor establishment. Eyes were collected 28 days after implantation, fixed, and embedded for histology and immunohistochemistry. All procedures complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center.

Statistical Analysis

Data are presented as mean ± SD. Statistical analyses were performed using GraphPad Prism 8.0.2. Comparisons were made using unpaired two-tailed Student's t-tests or one-way ANOVA, as appropriate. Significance levels are indicated in the figures as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).

Results

HLB-0532259 Induces Potent MYCN Degradation in RB Cells

HLB-0532259 is a PROTAC molecule designed to degrade MYCN by engaging AURKA as its binding interface and recruiting an E3 ligase to activate the ubiquitin–proteasome pathway (Fig. 1A). After 48 hours of HLB-0532259 treatment, both AURKA and MYCN protein levels decreased in a dose-dependent manner in Y79 and WERI-Rb1 cells (Figs. 1B, 1D). MYCN protein levels in Y79 cells were significantly reduced at 10 nM and were almost undetectable at 500 nM. WERI-Rb1 cells showed a similar dose response but with less pronounced degradation. Quantitative degradation-curve analysis showed a DC₅₀ of 8.13 nM for MYCN in Y79 cells and 6.97 nM in WERI-Rb1 cells (Figs. 1C, 1E). Consistently, HLB-0532259 reduced cell viability in a dose-dependent manner after 48 hours, with IC₅₀ values of 142.9 nM for Y79 cells and 1425 nM for WERI-Rb1 cells (Figs. 1F, 1G). Time-course studies further demonstrated that MYCN reduction in Y79 cells was evident as early as 12 hours after 100 nM HLB-0532259 exposure, whereas WERI-Rb1 cells exhibited a slower but still time-dependent decline (Figs. 1H, 1I).

Figure 1.

HLB-0532259 induces dose- and time-dependent degradation of MYCN in retinoblastoma cells. (A) Schematic illustration of the PROTAC molecule HLB-0532259. (B, D) Western blot analysis showing dose-dependent reduction of MYCN and AURKA protein levels in Y79 and WERI-Rb1 cells after 48 hours treatment with HLB-0532259. (C, E) Quantification of MYCN degradation curves in Y79 and WERI-Rb1 cells. (F, G) Cell viability assays demonstrating dose-dependent growth inhibition after 48 hours of HLB-0532259 treatment. (H, I) Time-course Western blot analysis showing progressive MYCN degradation after treatment with 100 nM HLB-0532259.

Temporal Transcriptome Analysis After MYCN Degradation

RNA-seq was performed to characterize the downstream transcriptional changes following MYCN degradation. Heatmaps revealed the correlation patterns among the 0-, 12-, 24-, and 48-hour groups (Fig. 2A). Differential expression analysis revealed the greatest number of differentially expressed genes (DEGs) at 12 hours (n = 4569), comprising 2831 upregulated and 1733 downregulated genes. This was followed by a decrease at 24 hours (2376 DEGs) and 48 hours (1658 DEGs), indicating the strongest cellular response at the early time point (Figs. 2B, 2C). Time-dependent clustering (Mfuzz) identified 12 distinct expression clusters with characteristic temporal patterns (Fig. 2D). Notably, cluster six DEGs displayed peak expression at 12 hours, and cluster 9 DEGs were upregulated at 48 hours. Pathway enrichment of these clusters showed significant involvement of apoptosis, cellular senescence, ferroptosis, necroptosis, and p53-related signaling (Figs. 2E, 2F). Among these processes, genes associated with cellular senescence displayed the most consistent and coordinated changes, prompting further investigation of senescence induction in RB cells.

Figure 2.

Temporal transcriptomic analysis reveals dynamic gene expression changes after MYCN degradation. (A) Heatmap showing sample correlations among control (0 hours) and HLB-0532259–treated groups (12, 24, and 48 hours). (B) Volcano plots showing DEGs at each time point. (C) Quantification of DEGs at each time point, showing the largest transcriptional response at 12 hours. (D) Time-series clustering of DEGs using Mfuzz analysis, identifying 12 distinct gene expression clusters with characteristic temporal patterns. (E) Pathway enrichment analysis of cluster 6 genes. (F) Pathway enrichment analysis of cluster 9 genes.

MYCN Degradation Induces Senescence via p53–p21 Signaling

Dynamic analysis of senescence-related genes revealed a staged program after treatment with HLB-0532259 (Fig. 3A). At 12 hours, the p53–p21 axis (TP53, CDKN1A) was rapidly activated, whereas DNA-repair regulators (RAD50, NBN) and PRC components (RING1, SUZ12) were downregulated, suggesting early proliferative arrest, impaired DNA repair, and release of epigenetic repression of senescence genes. From 24 to 48 hours, further induction of p53–p21 signaling and broad downregulation of histone genes indicated sustained cell-cycle arrest and progression toward irreversible senescence. Cell-cycle regulators E2F2 and CCNA2 showed transient early elevation followed by marked reduction, consistent with a transition from residual proliferation to complete arrest. Western blot validation confirmed these transcriptomic trends (Figs. 3B, 3C): SUZ12 protein was markedly reduced, supporting its role in chromatin remodeling during senescence initiation; and both p53 and p21 were strongly upregulated, identifying the p53–p21 pathway as a key effector of MYCN-degradation–induced senescence. The γ-H2AX accumulated at 24–48 hours, indicating sustained DNA damage. Although total NF-κB p65 levels declined, immunofluorescence demonstrated nuclear translocation beginning at 12 hours, accompanied by reduced cytoplasmic IκBα (Fig. 3D), suggesting activation of NF-κB signaling during late-stage senescence. In addition, EdU incorporation assays demonstrated a significant reduction in the proportion of EdU-positive cells following HLB-0532259 treatment (Fig. 3E), indicating marked suppression of DNA synthesis and proliferative activity. Consistently, soft agar colony formation assays further showed that HLB-0532259-treated cells exhibited a reduction in clonogenic capacity compared with control cells (Fig. 3F).

Figure 3.

MYCN degradation induces cellular senescence through activation of the p53–p21 pathway. (A) Heatmap showing temporal expression patterns of senescence-associated genes following HLB-0532259 treatment. (B, C) Western blot validation of key proteins involved in senescence signaling. (D) Immunofluorescence analysis of NF-κB signaling. (E) EdU incorporation assay in control and HLB-0532259-treated cells. (F) Soft agar colony formation assay in control and HLB-0532259-treated cells.

Proteomic Changes After MYCN Degradation

To complement transcriptomic findings, proteomic analysis was performed on 0-hour and 48-hour samples. PCA demonstrated clear separation between the two groups (Fig. 4A). Heatmap analysis identified 196 differentially expressed proteins, including 67 upregulated and 129 downregulated proteins at 48 hours (Fig. 4B). Senescence-associated secretory phenotype (SASP) proteins and DNA-damage-related factors—including TGFBI, ERCC4, and SET—were prominently upregulated, while oncogenic or MYCN-associated regulators such as HMGA1, TRAF6, and MIF were significantly downregulated (Fig. 4C). Western blotting further confirmed robust downregulation of HMGA1 and TRAF6 (Fig. 4D), supporting their involvement as MYCN-dependent effectors suppressed by HLB-0532259. Given the observed dissociation between TRAF6 downregulation and NF-κB activation, we further explored the involvement of ATM-dependent signaling. Pharmacological inhibition of ATM partially restored IκBα expression in HLB-0532259–treated cells (Fig. 4E), suggesting that NF-κB activation may, at least in part, be mediated through an ATM-dependent pathway under conditions of MYCN degradation. To further validate the induction of a senescence-associated secretory phenotype, additional SASP-related proteins, including GDF15, IGFBP3, and SERPINA1, were assessed by Western blot analysis and were found to be increased after HLB-0532259 treatment (Fig. 4F).

Figure 4.

Proteomic profiling identifies MYCN-dependent downstream effectors suppressed by HLB-0532259. (A) Principal component analysis (PCA) showing clear separation between control (0-hour) and HLB-0532259–treated (48-hour) samples. (B) Heatmap showing differentially expressed proteins identified by quantitative proteomics. (C) Selected differentially expressed proteins associated with senescence, DNA damage response, and MYCN signaling. (D) Western blot validation confirming decreased expression of HMGA1 and TRAF6 after HLB-0532259 treatment. (E) Analysis of NF-κB related signaling following ATM inhibition in HLB-0532259–treated cells. (F) Western blot analysis of SASP-associated proteins including GDF15, IGFBP3, and SERPINA1.

HLB-0532259 Efficacy and Senescence Pathway Activation in Orthotopic Xenografts

To evaluate the therapeutic efficacy, an orthotopic Y79 xenograft model was established. Mice received intravitreal HLB-0532259 on day 14, and eyes were harvested on day 28 (Fig. 5A). Gross examination revealed marked reduction of leukocoria in treated mice (Fig. 5B). Histology confirmed significantly decreased intraocular tumor burden following treatment (Figs. 5B, 5D). High-magnification examination demonstrated decreased tumor cellularity, disrupted tumor architecture, and sparsely distributed residual tumor cells within treated eyes (Supplementary Fig. S1). Immunohistochemistry demonstrated reduced Ki-67 and MYCN-positive cells in treated tumors (Figs. 5C–F). To further characterize target engagement over time in vivo, MYCN protein levels were assessed in tumor tissues at multiple time points after intravitreal administration. Western blot analysis revealed a marked reduction of MYCN expression at day 16, which remained consistently suppressed through day 28 (Fig. 5J), indicating sustained in vivo pharmacodynamic inhibition. Immunofluorescence further showed decreased cytoplasmic IκBα, increased nuclear NF-κB p65, and elevated p53 and p21 positivity within tumor cells (Figs. 5G, 5H), consistent with activation of senescence-associated pathways. The γ-H2AX staining revealed enhanced DNA damage in the treatment group (Fig. 5I), supporting the in vitro findings.

Figure 5.

HLB-0532259 suppresses tumor growth and activates senescence pathways in orthotopic retinoblastoma xenografts. (A) Experimental design of the orthotopic Y79 xenograft model and treatment schedule. (B) Representative images showing reduced leukocoria and tumor burden in treated mice compared with controls. (C–F) Immunohistochemical staining of tumor sections showing decreased MYCN and Ki-67 expression in the treatment group. (G, H) Immunofluorescence analysis demonstrating reduced cytoplasmic IκBα, increased nuclear NF-κB p65, and elevated p53 and p21 expression in tumor cells following HLB-0532259 treatment. (I) The γ-H2AX staining indicating increased DNA damage in treated tumors. (J) Western blot analysis of MYCN protein levels in tumor tissues at multiple time points after treatment. T, tumor; D, day; Tx, treatment.

Discussion

Amplification of MYCN is an important oncogenic driver in a subset of aggressive RB. However, targeting MYCN directly remains challenging due to the lack of a traditional small-molecule binding pocket. In this study, we evaluated the therapeutic potential of the PROTAC molecule HLB-0532259 in RB. HLB-0532259 treatment induced dose-dependent degradation of MYCN in RB cells and significantly reduced cell viability. Transcriptomic and proteomic analyses revealed that MYCN loss triggered broad transcriptional and protein changes associated with cellular senescence, with prominent activation of the p53–p21 pathway and induction of DNA-damage–related signaling. Furthermore, HLB-0532259 markedly suppressed tumor growth in an orthotopic xenograft model, which was accompanied by increased p53 and p21 expression in tumor tissues.

Under normal developmental conditions, N-Myc is characterized by an extremely short half-life, being rapidly targeted for proteasomal degradation via the ubiquitin-proteasome system.¹⁹ In MYCN^A tumors, however, binding of AURKA stabilizes N-Myc and prevents its degradation.¹⁸ Because this stabilization is largely independent of AURKA kinase activity, conventional AURKA inhibitors have limited effects on N-Myc protein stability.²⁰ HLB-0532259 is a heterobifunctional PROTAC molecule designed to target the AURKA–N-Myc complex. It contains an AURKA-binding ligand derived from the CDK4/6 inhibitor ribociclib, linked to a thalidomide-based cereblon (CRBN) recruiter that engages the CRL4^CRBN E3 ligase complex.¹⁸ In the present study, HLB-0532259 induced efficient MYCN degradation in both Y79 and WERI-Rb1 cells. Interestingly, the two cell lines showed markedly different phenotypic responses, with Y79 cells displaying nearly tenfold greater sensitivity in viability assays. This discrepancy suggests that the biological consequences of MYCN loss differ between the two cell lines. WERI-Rb1 cells exhibit a relatively differentiated phenotype with higher expression of mature retinal markers,²¹ whereas Y79 cells are less differentiated and display a highly proliferative profile.²² Such aggressive tumor cells are often considered to be in a state of oncogene addiction, in which cell survival depends heavily on MYCN-driven transcriptional programs. By comparison, the more differentiated WERI-Rb1 cells may partially rely on alternative regulatory networks for survival, thereby reducing their dependence on the AURKA–MYCN axis.

Degradation of MYC family proteins has previously been shown to induce cellular senescence.²³ In this study, HLB-0532259–mediated MYCN degradation triggered a rapid transcriptional reprogramming toward cellular senescence. The peak number of differentially expressed genes at 12 hours suggests that acute MYCN loss initiates an immediate transcriptional response. Notably, this early response was characterized by robust activation of the p53–p21 signaling axis, a central pathway known to drive therapy-induced senescence in many tumor types. At the same time, several DNA repair regulators, including RAD50 and NBN, were downregulated, accompanied by accumulation of γ-H2AX. Previous studies have suggested that MYC family proteins participate in the regulation of DNA replication and repair, and their loss can lead to replication stress and genomic instability.^24,25 Our findings are consistent with this concept and suggest that MYCN degradation may promote RB senescence partly through the accumulation of DNA damage. In addition, the significant reduction of polycomb repressive complex components (RING1, SUZ12) implies that MYCN may contribute to maintaining an epigenetically repressed. SUZ12 has been reported to be upregulated in RB and to promote tumor progression through enhanced H3K27me3-mediated transcriptional repression.²⁶

As the molecular program progressed from 24 to 48 hours, these early transcriptional changes evolved into a stable senescence phenotype. The widespread downregulation of histone genes and cell-cycle regulators such as E2F2 and CCNA2 is consistent with the establishment of irreversible cell-cycle arrest.^27,28 Proteomic profiling further supported this transition by revealing increased expression of SASP factors, including TGFBI, together with suppression of several MYCN downstream effectors such as HMGA1, TRAF6, and MIF. HMGA1 is an architectural transcription factor that has been identified as a direct transcriptional target of MYCN.²⁹ Its reduction after HLB-0532259 treatment, confirmed by Western blotting, therefore likely reflects the collapse of MYCN-dependent chromatin regulation. TRAF6 functions as a key signaling adaptor involved in NF-κB activation and inflammatory signaling pathways.³⁰ Interestingly, despite the reduction of TRAF6, activation of NF-κB signaling was still observed in treated cells. This paradox suggests that in the context of HLB-0532259-induced senescence, NF-κB activation may bypass TRAF6-dependent signaling. Instead, NF-κB activity may be sustained by persistent DNA damage responses, which are known to activate NF-κB through ATM-dependent pathways and promote the development of the SASP.³¹

Our in vivo results further highlight the therapeutic potential of pharmacological MYCN degradation in RB. Intravitreal administration of HLB-0532259 significantly suppressed intraocular tumor growth in orthotopic xenograft models. Because intravitreal injection enables high intraocular drug concentrations while limiting systemic toxicity,³² these findings support the feasibility of locally targeting MYCN in RB. Notably, reduced MYCN expression has been associated with a well-differentiated cavitary subtype of RB and more favorable clinical outcomes.³³ This clinical correlation is consistent with our results and suggests that MYCN suppression may promote a less aggressive tumor phenotype. However, MYCN-driven transcriptional programs are sustained by multiple cooperating regulatory layers, including BET bromodomain proteins, CDK7-mediated transcriptional control, and the miR-17-92 cluster.^6,34,35 In addition, evidence from neuroblastoma suggests that MYCN inhibition may induce initial tumor regression, whereas tumors can later adapt and partially bypass MYCN dependency, indicating that oncogene addiction is dynamic.³⁶ Collectively, these findings support pharmacological MYCN degradation as a promising therapeutic strategy in RB and further suggest that combination strategies targeting complementary MYCN-associated regulatory networks may enhance therapeutic efficacy and durability.

This study has several limitations. First, although our data demonstrate that HLB-0532259 induces MYCN degradation and triggers a senescence-associated transcriptional program, the downstream molecular networks linking MYCN loss to DNA damage accumulation and senescence remain incompletely defined. Further studies will be required to dissect the precise signaling pathways involved. Second, although intravitreal administration showed promising antitumor efficacy in vivo, the long-term pharmacokinetics, retinal safety, and potential off-target effects of HLB-0532259 require further evaluation. Third, the in vivo experiments were conducted in BALB/c nude mice, which lack functional T cell–mediated immunity. Therefore the observed antitumor effects do not fully account for the contribution of immune surveillance.

In summary, targeted MYCN degradation by the PROTAC molecule HLB-0532259 effectively suppresses RB growth both in vitro and in vivo. MYCN loss induces rapid transcriptional reprogramming characterized by activation of the p53–p21 pathway, accumulation of DNA damage, and development of a senescence-associated cellular state. Intravitreal delivery of HLB-0532259 significantly reduced tumor burden in orthotopic xenograft models, highlighting the potential of local MYCN-targeted therapy for RB.