Research ArticleOncology
Open Access | 10.1172/JCI176655
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
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1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
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1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
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1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Cai, H. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Shen, Z. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
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1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
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1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Zhang, J. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Zhang, X. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Bing, S. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Wang, J. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Shao, X. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Cao, J. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Yang, B. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by He, Q. in: PubMed | Google Scholar
1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
2Nanhu Brain-Computer Interface Institute, Hangzhou, China.
3Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
4Engineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou, China.
5School of Medicine, Hangzhou City University, Hangzhou, China.
Address correspondence to: Meidan Ying or Qiaojun He, Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China. Phone: 86.571.88208401; Email: Mying@zju.edu.cn (MY); Qiaojunhe@zju.edu.cn (QH).
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Find articles by Ying, M. in: PubMed | Google Scholar
Authorship note: SX and PC have been designated as co–first authors and contributed equally to this work.
Published June 10, 2025 - More info
Published in Volume 135, Issue 14 on July 15, 2025The N-Myc gene MYCN amplification accounts for the most common genetic aberration in neuroblastoma and strongly predicts the aggressive progression and poor clinical prognosis. However, clinically effective therapies that directly target N-Myc activity are limited. N-Myc is a transcription factor, and its stability is tightly controlled by ubiquitination-dependent proteasomal degradation. Here, we discovered that Kelch-like protein 37 (KLHL37) played a crucial role in enhancing the protein stability of N-Myc in neuroblastoma. KLHL37 directly interacted with N-Myc to disrupt N-Myc–FBXW7 interaction, thereby stabilizing N-Myc and enabling tumor progression. Suppressing KLHL37 effectively induced the degradation of N-Myc and had a profound inhibitory effect on the growth of MYCN-amplified neuroblastoma. Notably, we identified RTA-408 as an inhibitor of KLHL37 to disrupt the KLHL37–N-Myc complex, promoting the degradation of N-Myc and suppressing neuroblastoma in vivo and in vitro. Moreover, we elucidated the therapeutic potential of RTA-408 for neuroblastoma using patient-derived neuroblastoma cell and patient-derived xenograft tumor models. RTA408’s antitumor effects may not occur exclusively via KLHL37, and specific KLHL37 inhibitors are expected to be developed in the future. These findings not only uncover the biological function of KLHL37 in regulating N-Myc stability, but also indicate that KLHL37 inhibition is a promising therapeutic regimen for neuroblastoma, especially in patients with MYCN-amplified tumors.
IntroductionNeuroblastoma, derived from the peripheral sympathetic nervous system, is the most common extracranial solid tumor in children and accounts for 15% of pediatric tumor–associated deaths (1). Despite the standard treatments of surgical excision, radiotherapy, and chemotherapy, the prognosis for patients with high-risk neuroblastoma remains poor, with a 5-year survival rate of less than 50% (2–5). N-Myc gene (MYCN) amplification occurs in approximately 20% of patients with neuroblastoma, and all patients with MYCN-amplified tumors are classified as high risk with an unfavorable outcome (5–7).
Amplification of the MYCN oncogene leads to high expression levels and functional activation of N-Myc protein, which is a well-known oncogenic transcription factor. N-Myc is an important regulator that activates genes responsible for self-renewal, proliferation, and metastasis, while repressing genes involved in cell-cycle arrest, differentiation, and apoptosis (8–11). Transgenic overexpression of the N-Myc gene in mice and zebrafish induces tumors with characteristics similar to those of human neuroblastoma (12), whereas inhibition of N-Myc expression significantly arrests the growth of neuroblastoma (13). These insights suggest that efficient inhibition of N-Myc is supposed to achieve therapeutic benefits for patients with neuroblastoma. To date, many efforts have been made to develop targeted strategies against N-Myc, with inhibition targets including CDK7 (9), BRD4 (14), Aurora-A (15, 16), PLK1 (17), and ALDH18A1 (18). However, these strategies remain clinically unavailable in the treatment of neuroblastoma. Therefore, it remains urgent to elucidate the critical regulatory mechanisms of N-Myc and develop alternative efficient strategies that target N-Myc function.
The Kelch-like gene family (KLHL) proteins comprise 42 members that function as substrate adaptors in the Cullin 3–scaffold complex and regulate the ubiquitination process of substrate proteins, which have functions in the pathogenesis of various human diseases including cancer (19) Kelch-like protein 37 (KLHL37), also known as ectodermal-neural cortex 1 (ENC1), was first identified as being primarily expressed in the nervous system, where it participates in neuronal differentiation (20, 21), and studies have revealed that KLHL37 is upregulated in several tumors including colorectal carcinoma (22, 23) and breast cancer (24), and is associated with a poor patient prognosis (25, 26). Considering that both KLHL37 and N-Myc are associated with the nervous system, we wondered whether KLHL37 is associated with N-Myc and involved in the process of neuroblastoma.
In this study, we demonstrated that KLHL37 competitively interacted with N-Myc and released N-Myc from E3-mediated proteasomal degradation, leading to enhanced stabilization and aberrant activation of N-Myc, ultimately contributing to the malignant progression of neuroblastoma. Knockdown of KLHL37 or pharmacological inhibition of KLHL37 with the small molecule RTA-408 both markedly promoted the degradation of N-Myc and arrested neuroblastoma growth. These findings reveal the KLHL37/N-Myc axis as an essential oncogenic signaling axis underlying the progression of neuroblastoma. Moreover, we propose KLHL37 inhibitors as a selective therapeutic strategy for patients with MYCN-amplified neuroblastoma.
ResultsKLHL37 coordinates with N-Myc to promote neuroblastoma progression. To systematically uncover the relevance of KLHL family proteins to N-Myc, we first assessed the effect of the KLHL family on the proliferation of MYCN-amplified and non-MYCN-amplified neuroblastoma cells using an siRNA library targeting KLHLs. Our results revealed that selective inhibition of MYCN-amplified cells, but not non-MYCN-amplified cells, occurred upon silencing of KLHL12, KLHL30, and KLHL37 (Figure 1A). These observations suggest that these 3 KLHLs may have potential biological roles in neuroblastoma and may be associated with N-Myc. Moreover, we found that KLHL12 and KLHL37 had higher expression levels in tumor tissues compared with levels in adjacent tissues, whereas KLHL30 did not (Figure 1B). According to the transcriptomics data from patients with neuroblastoma (27), KLHL37 was observed to be significantly upregulated in patients with stage 4 disease, which is associated with a poor prognosis. By contrast, KLHL12 was expressed a low levels in patients with stage 4 disease, and KLHL30 did not exhibit significant changes across stages 1–4 (Figure 1C). Subsequent Kaplan-Meier analysis revealed that high expression of KLHL37, rather than KLHL12 or KLHL30, was significantly associated with poor overall survival (OS) of patients with neuroblastoma (Figure 1D). These findings suggest that KLHL37 may play a crucial role in the progression of neuroblastoma.
KLHL37 coordinates with N-Myc to promote neuroblastoma progression. (A) MYCN-amplified (MYCN-Amp) or non-MYCN-amplified (MYCN-Non) cells were transfected for 72 hours with a siRNA library specifically targeting KLHL proteins. The effect of the siRNAs on cell proliferation was examined and visually presented as a heatmap. SRB, sulforhodamine B. (B) KLHL37 expression in tumor (n = 59) or adjacent (n = 8) tissue was analyzed. FPKM, fragments per kilobase of transcript per million mapped reads. (C) The correlation between expression levels of KLHL12, KLHL30, and KLHL37 and clinical progression staging of patients with neuroblastoma was analyzed in comparison with the GEO GSE120572 cohort. The numbers of patients with stage 1, n = 54; stage 2, n = 67; stage 3, n = 58; stage 4, n = 168; stage 4S, n = 46, respectively. INSS, International Neuroblastoma Staging System. (D) Kaplan-Meier survival curves for OS of neuroblastoma patients with high (n =138) or low (n = 138) KLHL12, KLHL30, and KLHL37 expression levels. (E) Analysis of KLHL37 expression in patients with MYCN-amplified (n = 55) or non-MYCN-amplified (n = 222) neuroblastoma. Data were derived from the GEO GSE85047 cohort. (F) Effect of KLHL37-Flag overexpression on the clonogenic capacity of tumor cells with high N-Myc expression (SH-SY5Y and RH30) or low N-Myc expression (RD and NCI-H1299). (G) GSEA of the correlation of MYCN target gene signature enrichment with high KLHL37 expression in the GEO GSE85047 cohort. NES, normalized enrichment score; WEI, gene set. (H) Analysis of the impact of high KLHL37 expression and MYCN amplification on the progression-free survival (PFS) or OS of the patients with neuroblastoma based on the GEO GSE85047 cohort. The numbers of patients in the 4 groups are 211, 11, 44, and 11, respectively. (I) Effect of KLHL37 overexpression on the colony-forming ability of NIH-3T3 cells exogenously overexpressing N-Myc or C-Myc. *P < 0.05, **P < 0.01 and ***P < 0.001, by unpaired, 2-tailed Student’s t test (B, E, and F) and 1-way ANOVA (C and I). Data represent the mean ± SEM, except in F and I where data represent the mean ± SD.
Encouraged by the selective inhibitory effect of siKLHL37 on MYCN-amplified cells, we hypothesized that KLHL37 may play a regulatory role in N-Myc. By analyzing GSE85047 cohort (28), we found that KLHL37 was significantly upregulated in patients with MYCN-amplified neuroblastoma (Figure 1E). Meanwhile, we observed that when KLHL37 was overexpressed in different cancer cells, it promoted the clonogenic ability of these cells, and this enhancement was particularly pronounced in cells with an N-Myc expression background, such as neuroblastoma SH-SY5Y and rhabdomyosarcoma RH30 cells (Figure 1F and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI176655DS1). Further gene set enrichment analysis (GSEA) revealed that patients with neuroblastoma who had high expression of KLHL37 showed activation of N-Myc target genes (Figure 1G). Notably, high expression of KLHL37 in patients with MYCN-amplified neuroblastoma was associated with a worse prognosis compared with those with low KLHL37 expression (Figure 1H). These results indicate the potential correlation between N-Myc and KLHL37.
In addition, we observed that high KLHL37 expression was also associated with poor outcomes for patients with non-MYCN-amplified neuroblastoma. Given that N-Myc is dominant in patients with MYCN-amplified neuroblastoma, whereas its highly homologous protein C-Myc plays a role in patients with non-MYCN-amplified disease (29–31), as also indicated by their specific expression and CRISPR sensitivity (Supplemental Figure 1, B and C), we suspected that KLHL37 might also have a regulatory effect on C-Myc that would explain the poor prognosis of patients with non-MYCN-amplified disease. Consistently, we found that KLHL37 could enhance the clonogenic ability of cells expressing either N-Myc or C-Myc, with a more pronounced effect observed in cells expressing N-Myc (Figure 1I). Taken together, our findings suggest that KLHL37 might play an important role in the malignant progression of neuroblastoma, especially in patients with MYCN-amplified disease.
Targeting KLHL37 inhibits the progression of MYCN-amplified neuroblastoma. We next investigated whether targeting KLHL37 could inhibit the progression of MYCN-amplified neuroblastoma. The results showed that depletion of KLHL37 via an shRNA led to dramatic inhibition of the clonogenic ability in SK-N-DZ and SK-N-BE(2) cells, which harbor amplification of the MYCN gene, whereas a modest inhibitory effect was observed in non-MYCN-amplified SK-N-SH and SK-N-AS cells (Figure 2A and Supplemental Figure 2A). Next, 2 shRNA-resistant KLHL37 constructs based on shRNA 1 and 2 sequences were introduced, and they obviously rescued the inhibition effect of shKLHL37 on colony formation (Figure 2B and Supplemental Figure 2, B and C). Besides, KLHL37 knockdown induced significant apoptosis of MYCN-amplified neuroblastoma cells (Figure 2C and Supplemental Figure 2D). Further histological staining results indicated that KLHL37 knockdown significantly reduced N-Myc protein levels, inhibited cell proliferation, and induced cell apoptosis (Figure 2D and Supplemental Figure 2E). These findings indicate that targeting KLHL37 could inhibit the colony-forming ability and induce the death of MYCN-amplified neuroblastoma cells.
Targeting KLHL37 inhibits the progression of MYCN-amplified neuroblastoma. (A) Colony formation assay of neuroblastoma cells transduced with lentivirus-shKLHL37 (nos. 1 and 2). The colony-forming rate of shKLHL37 groups was calculated. (B) Overexpression of KLHL37-synonymous mutants, which were resistant to the knockdown effect of shKLHL37 no. 2, reversed the suppression of SK-N-DZ cell colony formation due to KLHL37 knockdown. (C) Effect of KLHL37 knockdown on apoptosis of SK-N-DZ, SK-N-BE(2), and SK-N-AS cells was determined by flow cytometry when cells were infected with shKLHL37 lentivirus for 5 days. (D) Analysis of histological staining for N-Myc and markers of proliferation and apoptosis in SK-N-DZ cells. (E) Images of SK-N-BE(2) xenograft tumors were captured at the end of the experiment. (F) Tumor volumes were measured every 2 days, and tumor growth curves are shown as the mean ± SEM. (G) Tumor weights of SK-N-BE(2) xenografts. (H) GSEA plots show the correlation between the enrichment of MYCN downstream genes and KLHL37 depletion. (I) Heatmap of changes in expression of survival-related genes in the KLHL37 depletion group. min, minimum; max, maximum. (J) Histological staining for N-Myc protein and markers of proliferation and apoptosis in SK-N-BE(2) xenograft tumors. Scale bar: 100 μm. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA (A–D, G, and I), unpaired, 2-tailed Student’s t test (B) and 2-way ANOVA (F) Data represent the mean ± SD in A, B, C, D, G, and J.
We further assessed the in vivo tumorigenicity of MYCN-amplified neuroblastoma cells transfected with shKLHL37. The results demonstrated that KLHL37 depletion obviously reduced the rate of tumor formation (100% in control vs. 60% in both shKLHL37 nos. 1 and 2) (Figure 2E and Supplemental Figure 3A). The average volume of tumors in the control groups increased continuously, reaching an average tumor volume at the end of the experiment of 1,652.8 mm3 compared with 92.7 mm3 and 411.7 mm3 in shKLHL37 no. 1 and no. 2 groups (Figure 2F). Moreover, shKLHL37 significantly reduced tumor weights (Figure 2G), and showed notable therapeutic activity, as indicated by a relative tumor volume (RTV) treatment/control (T/C) value of 6.31% and 25.26% (determined by RTVTreatment/RTVControl × 100%) (Supplemental Table 1). Additionally, no apparent body weight loss was observed in the shKLHL37 groups (Supplemental Figure 3B). Overall, these findings demonstrate that targeting KLHL37 exerted a remarkable inhibitory effect on the growth of MYCN-amplified xenograft tumors. Further RNA-Seq analysis revealed that N-Myc downstream and prosurvival genes were inhibited in the shKLHL37 groups (Figure 2, H and I). Additionally, immunohistochemical analysis showed that KLHL37 knockdown depleted N-Myc expression concomitant with obvious intratumoral proliferation inhibition and apoptosis activation as quantified by N-Myc, Ki67 and cleaved caspase 3 (c–caspase 3) staining (Figure 2J). In summary, these results show that KLHL37 might be a promising therapeutic target for MYCN-amplified neuroblastoma.
KLHL37 stabilizes N-Myc protein by interfering with its ubiquitination process. To understand the molecular process of the regulatory effect of KLHL37 on MYCN-amplified neuroblastoma, we wonder whether KLHL37 could directly regulate N-Myc protein. The results showed that KLHL37 knockdown led to a dramatic reduction of N-Myc protein levels in MYCN-amplified tumors both in vitro and in vivo (Figure 3, A and B). In addition, shKLHL37 also induced a modest reduction of C-Myc in non-MYCN-amplified cells (Figure 3A). Correspondingly, KLHL37 overexpression resulted in a remarkable upregulation of endogenous N-Myc protein levels and a moderate effect on C-Myc protein levels in cancer cells (Figure 3C and Supplemental Figure 4A). Similar phenomena were observed in exogenous N-Myc and C-Myc proteins using an isogenic cell model (Figure 3, D and E, and Supplemental Figure 4B). Notably, overexpression of shRNA-resistant KLHL37 successfully rescued the downregulation of N-Myc levels caused by shKLHL37 (Figure 3F and Supplemental Figure 4C). Altogether, these results demonstrate that KLHL37 was capable of regulating Myc protein level.
KLHL37 stabilizes N-Myc protein by interfering with its ubiquitination process. (A) Effect of KLHL37 knockdown on endogenous N-Myc protein expression. (B) KLHL37 depletion efficiency and N-Myc and C-Myc protein expression changes in SK-N-BE(2)-derived xenograft tumors were detected using immunoblotting. (C) Effect of KLHL37 overexpression on endogenous N-Myc protein levels in neuroblastoma cells. (D) Effect of KLHL37 knockdown on exogenously expressed N-Myc and C-Myc in RD cells. (E) Effect of KLHL37 overexpression on exogenous N-Myc protein levels in NIH-3T3 cells. (F) Overexpression of KLHL37-synonymous mutants, which were resistant to the knockdown effect of shKLHL37 no. 2, rescued the downregulation of N-Myc protein levels due to KLHL37 knockdown. (G) Effect of the proteasome inhibitor MG132 on the decline in N-Myc protein expression induced by knockdown of KLHL37. Cells were infected with lentivirus-shKLHL37 or control shRNA for 3 day and then treated with MG132 (10 μM) for 8 hours. (H) Effect of KLHL37 overexpression on the degradation rate of N-Myc protein. HEK-293T cells were infected with lentivirus to express N-Myc protein and then transfected for 48 hours with plasmids to overexpress KLHL37. Before harvesting, the cells were treated with cycloheximide (CHX) (10 μg/mL) for the indicated durations. (I) Effect of KLHL37 overexpression on the ubiquitination of N-Myc protein in the cell system. HEK-293T cells with stable expression of N-Myc-HA were transfected with plasmids to overexpress KLHL37 and His-Ub for 48 hours. Then cells were treated with MG132 (10 μM) for 8 hours before being harvested. (J) Effect of KLHL37 on the ubiquitination of N-Myc in vitro with the RRL system. (K) Effect of KLHL37 overexpression on the endogenous ubiquitination of N-Myc. CHP-126 cells were transfected for 48 hours with plasmids to overexpress KLHL37. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA (G) and 2-way ANOVA (H). Data represent the mean ± SD in G and H.
To gain insights into the reason why KLHL37 could regulate N-Myc protein levels, we first analyzed the mRNA levels of MYCN. We found that N-Myc mRNA levels showed no significant change upon KLHL37 overexpression (Supplemental Figure 4D), suggesting that KLHL37 might be involved in the posttranslational regulation process of N-Myc. Given that N-Myc is a short-lived protein that undergoes rapid degradation through the ubiquitin proteasome system (15, 17), we speculated that KLHL37 might potentially cause an imbalance in the ubiquitination-mediated degradation of N-Myc. The results showed that the decline in N-Myc levels caused by shKLHL37 was restored by treatment with the proteasome inhibitor MG132 (Figure 3G). Meanwhile, overexpressed KLHL37 markedly prolonged the degradation half-life of N-Myc protein (Figure 3H). We further performed ubiquitination assays on HEK-293T cells and found that ubiquitination of N-Myc was obviously suppressed by KLHL37 overexpression (Figure 3I). Furthermore, we constructed an in vitro model based on recombinant KLHL37 and N-Myc proteins, using rabbit reticulocyte lysate (RRL) as a tool for enabling ubiquitination modification, and found that KLHL37 was capable of removing the ubiquitin chains from N-Myc (Figure 3J). A similar result was observed on endogenous N-Myc ubiquitination in neuroblastoma cells (Figure 3K). Collectively, these findings demonstrate that KLHL37 could increase the stability of N-Myc by blocking its ubiquitination degradation process.
KLHL37 competes with the E3 enzyme to bind N-Myc and prevents its degradation. To elucidate the mechanism underlying the regulation of N-Myc protein stability by KLHL37, we first investigated the potential direct interaction between KLHL37 and N-Myc. We found that KLHL37 interacted with exogenously overexpressed N-Myc (Figure 4A) and endogenous N-Myc by co-IP (Figure 4B). A proximity ligation assay (PLA) also indicated their in situ interaction in neuroblastoma cells (Figure 4C). Moreover, a direct interaction between recombinant KLHL37 and N-Myc proteins was further confirmed by co-IP (Figure 4D) and a microscale thermophoresis assay (Figure 4E). Besides N-Myc, we also observed the binding between C-Myc and KLHL37, which was weaker than that of N-Myc (Figure 4E and Supplemental Figure 5A). It is well known that the 6-repeat Kelch domain of the KLHLs family of proteins serves to form a pocket to capture substrate proteins (32, 33), thus we constructed KLHL37-ΔKelch and KLHL37-Kelch–repeat truncation mutants to clarify which region of KLHL37 is required for N-Myc binding (Supplemental Figure 5B). Co-IP results showed that deletion of the Kelch domain abolished the interaction between KLHL37 and N-Myc (Figure 4F), suggesting that N-Myc binds to the Kelch domain of KLHL37. Taken together, these findings uncover the interaction basis of the regulatory effect of KLHL37 on N-Myc.
KLHL37 competes with the E3 enzyme to bind N-Myc and prevents its degradation fate. (A) The interaction between exogenous KLHL37 and N-Myc was detected using an IP assay. HEK-293T cells were transfected for 48 hours with plasmids overexpressing KLHL37-HA and N-Myc–Flag. (B) Cells were transfected for 48 hours with plasmids to overexpress KLHL37. KLHL37-Flag was enriched using anti-Flag resin, and then N-Myc signal was determined by immunoblotting. (C) A PLA was performed to detect the in situ interaction between KLHL37 and N-Myc proteins in SK-N-BE(2) cells. Scale bar: 10 μm. (D) Direct interaction between KLHL37-Flag and GST–N-Myc recombinant proteins in a cell-free system. KLHL37 bound to N-Myc was detected by immunoblotting, and other signals were visualized as Coomassie brilliant blue staining. (E) MST was performed to detect the interaction affinity of recombinant KLHL37 protein and recombinant GFP–N-Myc (rGFP–N-Myc) and GFP–C-Myc (rGFP–C-Myc) proteins. Mean ± SD.(F) HEK293T cells were transfected with different plasmids to overexpress N-Myc, WT KLHL37, and the truncated form of KLHL37 (Kelch repeats) for 48 hours. (G) Competition between KLHL37 and FBXW7 in binding to N-Myc proteins. GST or GST–N-Myc recombinant proteins were preincubated with KLHL37-His recombinant protein at 25°C for 4 hours. They were then incubated with cell extracts from HEK-293T cells overexpressing FBXW7-HA at 4°C for 12 hours. (H) Cells were transfected with plasmids to overexpress KLHL37 for 48 hours, and the interaction between endogenous N-Myc and FBXW7 was determined by co-IP and immunoblotting. (I) Effect of KLHL37 on the in vitro ubiquitination of N-Myc in a RRL cell-free system when recombinant FBXW7 protein was added to the system to promote N-Myc ubiquitination. (J) Interaction between KLHL37 and the N-Myc 1–89 aa segment. HEK-293T cells were transfected for 48 hours with plasmids to overexpress the indicated proteins, and the interaction was determined by immunoblotting after a co-IP assay.
It is established that KLHL family proteins act as E3 ligase adaptors, which bind to the scaffold protein Cullin 3 and in turn recruit Rbx1 and E2-Ub, thereby promoting the ubiquitination and degradation of substrate proteins (34–36). However, our findings showed that KLHL37 suppressed the degradation of N-Myc, which contrasts with the Cullin 3 E3 ligase function of other KLHLs. Accordingly, we hypothesized that the role of KLHL37 in regulating N-Myc might be independent of the KLHL–Cullin 3 E3 complex. To prove this, we deleted the 3-box region of KLHL37, which is crucial for binding Cullin 3 (32). The results showed that KLHL37 (Δ3-box) failed to interact with Cullin 3 a
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