Superenhancer activation of KLHDC8A drives glioma ciliation and hedgehog signaling
Derrick Lee, … , David R. Raleigh, Jeremy N. Rich
Derrick Lee, … , David R. Raleigh, Jeremy N. Rich
Published November 17, 2022
Citation Information: J Clin Invest. 2023;133(2):e163592. https://doi.org/10.1172/JCI163592.
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Research Article Oncology

Superenhancer activation of KLHDC8A drives glioma ciliation and hedgehog signaling

Abstract

Glioblastoma ranks among the most aggressive and lethal of all human cancers. Self-renewing, highly tumorigenic glioblastoma stem cells (GSCs) contribute to therapeutic resistance and maintain cellular heterogeneity. Here, we interrogated superenhancer landscapes of primary glioblastoma specimens and patient-derived GSCs, revealing a kelch domain–containing gene, specifically Kelch domain containing 8A (KLHDC8A) with a previously unknown function as an epigenetically driven oncogene. Targeting KLHDC8A decreased GSC proliferation and self-renewal, induced apoptosis, and impaired in vivo tumor growth. Transcription factor control circuitry analyses revealed that the master transcriptional regulator SOX2 stimulated KLHDC8A expression. Mechanistically, KLHDC8A bound chaperonin-containing TCP1 (CCT) to promote the assembly of primary cilia to activate hedgehog signaling. KLHDC8A expression correlated with Aurora B/C Kinase inhibitor activity, which induced primary cilia and hedgehog signaling. Combinatorial targeting of Aurora B/C kinase and hedgehog displayed augmented benefit against GSC proliferation. Collectively, superenhancer-based discovery revealed KLHDC8A as what we believe to be a novel molecular target of cancer stem cells that promotes ciliogenesis to activate the hedgehog pathway, offering insights into therapeutic vulnerabilities for glioblastoma treatment.

Authors

Derrick Lee, Ryan C. Gimple, Xujia Wu, Briana C. Prager, Zhixin Qiu, Qiulian Wu, Vikas Daggubati, Aruljothi Mariappan, Jay Gopalakrishnan, Matthew R. Sarkisian, David R. Raleigh, Jeremy N. Rich

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Figure 3

KLHDC8A expression is driven by SOX2 and a GSC superenhancer in GSCs.

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KLHDC8A expression is driven by SOX2 and a GSC superenhancer in GSCs. (A...
(A) KLHDC8A mRNA expression was measured in 3 matched pairs of GSCs and DGCs by qPCR analysis. n = 3. Quantitative data from 3 independent experiments are shown as mean ± SD. Statistical analysis was performed using Student’s t-test with the Holm-Šidák multiple test correction. (B) Protein levels of KLHDC8A were measured by immunoblot following KLHDC8A knockdown. SOX2 was used as the stemness marker. β-Actin was used as the loading control. (C) H3K27ac signals at the KLHDC8A superenhancer region in 3 pairs of GSCs and DGCs (MGG4, MGG6, and MGG8). SOX2 and OLIG2 ChIP-Seq signals are shown at the superenhancer region of MGG8. (D) Correlation of mRNA expression between KLHDC8A, OLIG2, and SOX2 in the TCGA HG-U133A glioblastoma data set. Numbers indicated the R value of Spearman correlation. (E) qPCR analysis of mRNA expression of SOX2 and KLHDC8A upon knockdown of SOX2. n = 3. Quantitative data from 3 independent experiments are shown as mean ± SD. Statistical analysis was performed using 2-way ANOVA with Dunnett’s multiple test correction. (F) qPCR analysis of mRNA expression of KLHDC8A following treatment with 2 concentrations of JQ1 (1.5 and 3 μM) for 24 hours. n = 3. Quantitative data from 3 independent experiments are shown as mean ± SD. Statistical analysis was performed using 2-way ANOVA with Dunnett’s multiple test correction. (G) Schematic displaying targeting of the KLHDC8A superenhancer region using dCas9-KRAB system with 5 nonoverlapping sgRNA targeting critical KLHDC8A superenhancer locus. (H) The mRNA expression of KLHDC8A in GSC23 and GSC3028 was measured by quantitative PCR. n = 4. Quantitative data from 4 independent experiments are shown as mean ± SD. Statistical analysis was performed using 2-way ANOVA with Dunnett’s multiple comparison. (I) Proliferation of GSCs measured by CellTiter-Glo assay in GSC23 and GSC3028 overexpressing dCas9-KRAB and 5 sgRNAs over a 6-day time course. n = 4. Quantitative data from 4 technical replicates are shown as mean ± SD. Statistical analysis was performed using 2-way ANOVA with Dunnett’s multiple test correction. *P < 0.05, **P <0.01, ***P < 0.001, ****P < 0.0001.