Targeting PUS7 suppresses tRNA pseudouridylation and glioblastoma tumorigenesis

The story started from a simple but unanswered question: what is the cellular function of pseudouridine, the most frequent epitranscriptomic modification, in cancer?

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Back to 2017, with growing evidences shown by us and others indicating that RNA modifications, e.g. m6A RNA modification, play a critical role in cancer development1, 2, we were wondering how pseudouridine as an abundant RNA modification could impact cancer, such as glioblastoma or GBM, the most aggressive primary brain tumor.

We first looked at several GBM databases for expressions of pseudouridine synthase (PUS) enzymes in patients and analyzed the association between PUS expression and patient survival. It attracted our attention that PUS7 is highly expressed in GBM patient samples compared to normal control samples and higher PUS7 expression level in GBM patients is associated with worse patient median survival. We immediately confirmed the elevated expression level of PUS7 in GBM patient brain tissues compared to non-tumor tissues. Moreover, we found that PUS7 is highly expressed in glioblastoma stem cells (GSCs) compared to normal neural stem cells (NSCs). These results together suggest that PUS7 could be an important regulator of GBM tumorigenesis. The differential expression of PUS7 in GSCs and NSCs suggests that targeting PUS7 may allow us to develop drugs that specifically targets GSCs but not NSCs, a topic of interest to us for years.

Next, we investigated the role of PUS7 in GSC self-renewal and tumorigenesis. We found that targeting PUS7 through shRNA-mediated knockdown or CRISPR/Cas9-mediated knockout sufficiently reduced the growth and self-renewal of GSC. Accordingly, suppression of PUS7 expression in GSC dramatically inhibited GSC tumorigenesis and extended the lifespan of tumor-bearing mice in an orthotopic patient-derived xenografts (PDX) model. Thus, we have provided a direct evidence showing that targeting PUS7 could suppress GSC-derived tumor progression.

Having demonstrated the efficacy of targeting PUS7 for glioblastoma, the next question is whether we can target PUS7 in a pharmacologically relevant manner, a question that is of great interest to the field of RNA modification in cancer. By applying a virtual screening to identify small molecules targeting PUS7 enzymatic activity followed by a secondary biochemical screening using the PUS activity as the readout, we discovered a small molecule compound that could efficiently suppress the enzymatic activity of PUS7 on pseudouridine modification. The inhibition of pseudouridine modification was confirmed in GSCs after the PUS7 inhibitor treatment. Moreover, the PUS7 inhibitor suppressed GSC growth in vitro and GBM tumorigenesis in vivo, indicating that pharmacological targeting of PUS7 can be an effective approach to inhibit GBM tumorigenesis.

Till now, the question of how PUS7 regulates GBM tumorigenesis is remained. To address this challenging question, we first identified pseudouridine modification profiles in small RNAs, including snRNAs and tRNAs, in which we observed apparent pseudouridine modification level decrease upon knockout of PUS7 in GSCs. Accordingly, we have detected a list of tRNAs with pseudouridine modification sites dependent on PUS7 by comparing the profile in control GSCs and in GSCs with PUS7 knockout. Interestingly, we did not detect a global translation change in GSCs upon PUS7 knockout. Instead, we saw PUS7 regulating translation in a more specific manner through altering the translation of its targeted tRNAs, with increased translation of targeted tRNAs upon PUS7 knockout in GSCs. In addition to small RNAs, we profiled the pseudouridine modification in other types of RNAs in GSC, including rRNA and mRNA with a list of PUS7 dependent sites identified in mRNA in GSC.

Next, we performed whole transcriptome analysis by RNA-seq and quantitative proteomics using the tandem mass tag (TMT) analysis. We found that key regulators (e.g. TYK2) were controlled by PUS7 at the protein level and impact important signaling pathways (e.g. IFN pathway) in GSCs. Importantly, PUS7 regulates the key regulators (e.g. TYK2) in GSC through codon-specific translational control via its targeted tRNAs. PUS7 in turn regulates GSC growth through TYK2-mediated IFN pathway.

In summary, we found that PUS7 is highly expressed in glioblastoma and is critical for GSC tumorigenesis. Mechanistically, pseudouridylation of PUS7-targeted tRNA regulates key regulators of GSCs by codon-specific translational control. Moreover, we identified small molecule inhibitors of PUS7 and provided preclinical evidence that targeting PUS7 could be a potential therapeutic strategy for treating glioblastoma.

This work was done through collaborations, with great efforts from all collaborative groups, despite the difficulty that we had to go through during the pandemic. We hope our work will be of interest to others and contribute to the advance of the field.

References

  1. Cui, Q. et al. m(6)A RNA Methylation Regulates the Self-Renewal and Tumorigenesis of Glioblastoma Stem Cells. Cell Rep 18, 2622-2634 (2017).
  2. Zhang, S. et al. m(6)A Demethylase ALKBH5 Maintains Tumorigenicity of Glioblastoma Stem-like Cells by Sustaining FOXM1 Expression and Cell Proliferation Program. Cancer Cell 31, 591-606 e596 (2017).

Yanhong Shi

Professor, Beckman Research Institute of City of Hope