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Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2 (SLAM-Seq)

ABSTRACT: The hallmark oncogene MYC drives the progression of most human tumors, but direct inhibition of MYC by small molecules has not yet entered the clinical testing phase. As a transcription factor, MYC is dependent on a variety of binding partners for its molecular oncogenic function. Here, we explored the possibility of targeting MYC via its interactome, through a genetic screen in pancreatic ductal adenoma carcinoma (PDAC) models. While several MYC-binding partners are important for cultured PDAC cells but dispensable for tumors in their natural environment in mice, the nuclear AAA-ATPases RUVBL1 and -2 are most essential among all MYC-binding partners in vivo. Induced degradation of RUVBL by the auxin-degron system leads to rapid arrest of PDAC cells in culture and complete regression of tumors in mice, which is preceded by immune cell infiltration. Mechanistically, RUVBL is required for MYC-mediated elongation of RNA polymerase II (RNAPII) and thus for the establishment of MYC dependent oncogenic gene expression patterns. Overall, three relevant observations emerged from our study. First, our study shows that tumor cell dependencies are strongly influenced by their environment and that genetic screens in cultured cells need to be carefully validated in vivo. Second, we have shown that the auxin-degron system can be applied in a PDAC model, allowing target validation and molecular analysis in living mice. Third, our study identifies RUVBL1 as a druggable vulnerability in MYC driven cancer.

ORGANISM(S): Mus musculus

PROVIDER: GSE216092 | GEO | 2024/06/01

REPOSITORIES: GEO

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Project description:Introduction Genome-wide association studies along with expression quantitative trait loci (eQTL) have identified hundreds of single nucleotide polymorphisms (SNPs) and their target genes in prostate cancer (PrCa). Although these genetic associations to PrCa have been widely reported, functional characterization of these risk loci remains challenging. Methods To screen for regulatory SNPs, we designed a library containing 9133 guide RNAs (gRNAs) to target 2,166 candidate SNP sites implicated in PrCa. We performed negative screening in dCas9-KRAB stable prostate cell lines and applied the RIGOR program to identify the essential SNPs for cell proliferation. We further characterized the regulatory role of a selected single nucleotide polymorphim (SNP, rs60464856) using luciferase reporter assay, ChIP-qPCR, and xCas9 base editing in prostate cells. Finally, we investigated the biological impact of the SNP-regulated gene RUVBL1 on cell proliferation and tumor growth via gene knockdown using in vitro and in vivo assays. Results From interference of 2,166 candidate SNPs via CRISPR interference screening, the RIGOR program identified 117 SNPs that could regulate genes to promote growth advantage in prostate cancer cell lines. Compared to unselected SNPs, the 117 candidates tended to reside near 5 kb flanking the transcription start sites (p = 0.01). To characterize the regulatory role of these SNPs, we selected one SNP (rs60464856) for detailed analysis. This SNP was covered by multiple gRNAs significantly depleted in the screening (FDR<0.05). Pooled SNP association analysis in the PRACTICAL cohort showed significantly higher PrCa risk for the G allele (pvalue=1.2E-16). eQTL analysis showed that the G allele is associated with an increased expression of RUVBL1 in multiple datasets. To further validate the CRISPR interference effect, we transfected a gRNA targeting the rs60464856 site in the dCas9 stable cell lines and observed significant inhibition of the RUVBL1 expression. We also applied the xCas9 adenine base editor to convert the rs60464856 A into G allele and observed an increased RUVBL1 expression in subclones carrying the rs60464856 G allele in prostate cell lines. To test if any protein showed allele-specific binding at rs60464856, we used SILAC-based DNA pull-down proteomics and observed that cohesin subunits (including SMC3) preferred the A allele. ChIP qPCR assays showed significant enrichment of CTCF and SMC3 signals at the rs60464856 site with preferential binding to the A allele. To evaluate the potential role of this locus in maintaining long-range chromatin structure, we analyzed a HiC dataset and found that the rs60464856 locus enriched consistent chromatin interactions in prostate cell lines. To determine the potential role of the rs60464856 target gene, we knocked down the RUVBL1 via shRNA and observed significant proliferation inhibition in prostate cell lines. We also tested PC3 cells with RUVBL1 knockdown in nude mice xenografts and observed reduced tumorigenesis. Gene set enrichment analysis showed that RUVBL1 expression was associated with the enrichment of cell cycle related pathways in both cell line and TCGA prostate cancer cohorts. Lastly, we showed that an increased RUVBL1 expression and its relevant pathway activation were associated with poor survival. Conclusion We applied the CRISPR interference screening at selected prostate cancer risk loci and identified over a hundred regulatory SNPs essential for prostate cell proliferation. Further analysis confirmed the important role of rs60464856 and its target gene RUVBL1 in prostate cell growth and tumorigenesis. To validate one of our candidate, we knocked down RUVBL1 gene in BPH1 and PC3 cell lines and observed the transcriptome alterations with RNA-seq

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Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2 (SLAM-Seq)

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