Interaction between DNA damage response, translation and apoptosome determines cancer susceptibility to TOP2 poisons

Abstract

Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We show that glioblastoma (GBM), the most malignant of all primary brain tumors in adults is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 impaired the induction of pro-apoptotic gene APAF1 following etoposide treatment, and led to resistance to this drug and doxorubicin. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from multiple cancers. KeywordsCRISPR, Glioblastoma (GBM), Topoisomerase II poisons (TOP2: etoposide, doxorubicin), DNA damage and repair response, H2AX (gamma H2AX phosphorylation) FANCB, Protein Synthesis, Ribosomal proteins subunits: (RPS11, 16, and 18), APAF1, Pro-(BID, CASPASE3/7) and anti-apoptotic effectors (BCL2).

Amino acyl tRNA synthetase inhibitors is therapeutics for gliomas at sub nanomolar concentrations

“…Briefly, as previously described (Awah et al, 2019), we obtained Brunello SgRNA library from Addgene ( 73179-LV), this library contains 70,000 sgRNA. 60,000 of which target the 20,000 genes at 3-4 guides per gene and 10,000 non-targeting controls.…”

“…At the end of 14 days, the cells were harvested and the genomic DNA from the DMSO and the etoposide treated groups were harvested using Zymo Research gDNA kit and the SgRNA were amplified by a unique set of barcodes, then using NextSeq, the SgRNA abundance was determined. The data was analyzed as described (Awah et al, 2019) using CRISPRAnalyzer (Winter et al, 2017). We used 8 statistical methods from the CRISPRAnalyzer to call a guide a hit using a very stringent p<0.001 and an FDR of 0.01.…”

“…AARS has been shown to be highly over-expressed in tumors such as glioblastoma (Awah et al, 2019;Kim et al, 2012), prostate cancer (Vellaichamy et al, 2009), and other tumors. Furthermore, various small molecules have been shown to specifically inhibit different types of amino acyl tRNA synthetases.…”

“…To repurpose a well-known DNA damaging agent, etoposide for glioblastoma, we performed a genomescale CRISPR knockout screen in a glioma cell line under etoposide selection (Awah et al 2019). It was found that the most enriched themes were genes in protein translation (Awah et al, 2019), whic h belonged to amino acyl tRNA synthetase and ribosomal protein subunits.…”

“…To repurpose a well-known DNA damaging agent, etoposide for glioblastoma, we performed a genomescale CRISPR knockout screen in a glioma cell line under etoposide selection (Awah et al 2019). It was found that the most enriched themes were genes in protein translation (Awah et al, 2019), whic h belonged to amino acyl tRNA synthetase and ribosomal protein subunits. In addition, using the genome-scale CRISPR, we also found that glioblastoma expresses high levels of AARS and inhibition of AARS with borrelidin and mupirocin specifically kills the glioma cell lines in-vitro and at a very low concentration.…”