Neuroblastoma is the most frequent extracranial solid tumor in children, arising from neural crest cells. While treatments like surgery, chemotherapy, radiotherapy, and immunotherapy exist, high-risk neuroblastoma, often characterized by MYCN amplification, remains challenging with high relapse rates. This necessitates the exploration of novel therapeutic strategies. Indisulam (E7070), a dual sulfonamide, has shown antitumor activity in various cancers by targeting multiple cell cycle checkpoints and downregulating cyclins and CDKs. However, its efficacy in neuroblastoma remained untested. Recent research unveiled indisulam's precise mechanism: it acts as a molecular glue, inducing a ternary complex between RBM39 (an RNA binding protein) and the E3 ubiquitin ligase DCAF15, leading to RBM39 degradation, aberrant RNA splicing, and ultimately, cell death. RBM39's involvement in splicing complexes and its role as a transcriptional coactivator suggest its targeting could have broad effects on cellular processes, including metabolism. This study aimed to investigate indisulam's efficacy in neuroblastoma models, exploring its mechanism of action, particularly concerning its impact on RNA splicing and metabolism, and its potential as a therapeutic for high-risk neuroblastoma.

Previous studies have demonstrated indisulam's ability to inhibit cell cycle progression through targeting multiple checkpoints in the G1 and G2 phases. It disrupts and downregulates key cell cycle regulators such as cyclin A, cyclin B, CDK2, and CDC2, often via p21/p53-dependent mechanisms. Preclinical studies showed promising antitumor effects in HCT116 xenografts, superior to 5-FU and Irinotecan, leading to phase I/II clinical trials in other advanced solid tumors. Although indisulam exhibited acceptable toxicity profiles, clinical responses were modest, and its efficacy in neuroblastoma was unexplored. The recent discovery of indisulam's mechanism—targeting RBM39 for degradation via DCAF15—provided a clearer understanding of its action and potential for targeted therapy. RBM39's involvement in pre-mRNA splicing and its association with splicing defects highlighted the possibility of targeting RNA processing as a therapeutic strategy. Furthermore, RBM39's role as a coactivator of transcription factors and its potential regulation of metabolism via the NF-κB/c-Myc pathway suggested broader effects on cellular function beyond cell cycle control.

This study employed a multifaceted approach to investigate indisulam's efficacy and mechanism in neuroblastoma. Initially, publicly available drug sensitivity data were analyzed to identify tumor types sensitive to indisulam, revealing neuroblastoma's high sensitivity. In vitro studies utilized neuroblastoma cell lines IMR-32 and KELLY, assessing growth inhibition and apoptosis using SRB assays, 3D spheroids, and caspase activation assays. To confirm RBM39 degradation, LC-MS-based global label-free proteomics was performed, followed by western blotting validation. RNA sequencing and SpliceFisher analysis were used to identify splicing errors following indisulam treatment. Integrative analysis of transcriptomic and proteomic data elucidated the impact of indisulam on cellular pathways, particularly those involved in cell cycle regulation and metabolism. Metabolic profiling via LC-MS and 13C isotope tracing (using glucose and glutamine) was conducted to identify metabolome perturbations and mitochondrial dysfunction. CRISPR-Cas9 technology was employed to generate DCAF15 knockout KELLY cells to investigate the role of DCAF15 in indisulam's mechanism. In vivo studies utilized IMR-32 xenograft and Th-MYCN transgenic mouse models to assess indisulam's efficacy in vivo. Tumor regression, survival rates, and pharmacodynamic markers (RBM39 levels, splicing changes, and metabolic alterations) were monitored. Finally, the role of MYCN amplification in determining sensitivity to indisulam was investigated using cell lines with and without MYCN amplification, and a Tet21 model with doxycycline-inducible MYCN expression.

The study revealed that indisulam effectively inhibited neuroblastoma growth and induced apoptosis in vitro in both IMR-32 and KELLY cell lines. Indisulam treatment resulted in a significant and selective DCAF15-dependent degradation of RBM39, leading to a substantial increase in RNA mis-splicing events (exon skipping and intron retention). Proteomics and transcriptomics data showed that indisulam's effects extended beyond RNA splicing, significantly impacting cell cycle and metabolic pathways. Metabolic profiling demonstrated perturbations in one-carbon metabolism and mitochondrial dysfunction. Notably, some metabolic changes were observed to be DCAF15-independent. In vivo studies demonstrated complete and sustained tumor regression in both IMR-32 xenograft and Th-MYCN transgenic mouse models following indisulam treatment, with in vivo confirmation of RBM39 loss, splicing errors, and metabolic alterations. Finally, MYCN amplification was identified as a significant determinant of sensitivity to indisulam, suggesting that high-risk MYCN-amplified neuroblastoma may be particularly responsive to this drug.

This study provides compelling evidence for indisulam as a promising therapeutic agent for high-risk neuroblastoma. The findings demonstrate that indisulam's efficacy stems from its dual targeting of RNA splicing and metabolism. The DCAF15-dependent degradation of RBM39, leading to widespread splicing errors and disruptions in cell cycle and metabolic pathways, effectively inhibits tumor growth. The observed complete and sustained tumor regression in vivo, even in a challenging MYCN-amplified model, strongly supports the clinical translation potential of indisulam. The identification of MYCN amplification as a predictor of sensitivity further enhances the drug's appeal for targeting high-risk neuroblastoma patients. The findings expand our understanding of RBM39's role in cancer biology and identify potential biomarkers (such as DCAF15 expression and specific metabolic changes) for predicting treatment response and monitoring treatment efficacy.

This study demonstrates that indisulam is a highly effective anti-cancer agent in neuroblastoma models, exhibiting complete tumor regression in vivo. Its mechanism involves DCAF15-dependent RBM39 degradation, leading to aberrant splicing, cell cycle disruption, and metabolic reprogramming. MYCN amplification correlates with increased sensitivity to indisulam. Given the pre-existing clinical data and the drug's tolerable toxicity profile, indisulam is a strong candidate for repurposing in clinical trials for high-risk neuroblastoma. Further research should focus on identifying additional predictive biomarkers and exploring the potential of combining indisulam with other therapies to further enhance its efficacy.

While the study provides strong preclinical evidence, further investigation is needed to fully understand the potential off-target effects of indisulam and the long-term consequences of its metabolic effects. The in vivo studies used specific mouse models, and the translatability of these findings to human patients needs to be confirmed in clinical trials. Additionally, the specific mechanisms by which MYCN amplification influences sensitivity to indisulam warrant further investigation. The sample sizes in some in vivo experiments could be considered relatively small, requiring larger scale studies to confirm the findings.