Alkylation Sensitivity Screens Reveal a Conserved Cross-species Functionome

Abstract

To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases UNG and MYH as well as MPG to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in E. coli and S. cerevisiae. The conserved biological processes across all three species composes an Alkylation Functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance.

UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase

“…These findings are consistent with a role for UBE3B in the cellular response to mitochondrial oxidative stress, because it was shown that TMZ treatment triggers a rapid onset of ROS (29). Similarly, it was shown that the Caenorhabditis elegans ortholog of UBE3B, oxi-1, is required for protein degradation via the UPS, especially under oxidative stress conditions and that deletion of the mouse Ube3b gene led to reduced viability and brain size (25).…”

“…In a recent synthetic lethal screen done by our laboratory using an siRNA library, the chemotherapy agent temozolomide (TMZ), and a glioblastoma multiforme cell line, we found that knockdown (KD) of UBE3B sensitized cells to TMZ treatment (29). These findings are consistent with a role for UBE3B in the cellular response to mitochondrial oxidative stress, because it was shown that TMZ treatment triggers a rapid onset of ROS (29).…”

Molecular Genetics of Secondary Glioblastoma

“…Accumulating evidence from numerous studies suggests that acquired treatment resistance following TMZ administration is multifactorial and rooted in transcriptional, metabolomic, genomic, and epigenomic changes that lead to this phenotype (60)(61)(62)(63)(64)(65)(66)(67).…”

A Role for Saccharomyces cerevisiae Tpa1 Protein in Direct Alkylation Repair

“…MMS Sensitivity of the mag1⌬tpa1⌬ Double Mutant Strain Is Not Due to Defective Protein Translation-Increased sensitivity to the alkylation agent MMS has also been reported to arise when a deficiency of proteins involved in the BER pathway is combined in the protein modification pathway (52). In other words, if Tpa1 were involved in the regulation of protein synthesis, then the mag1⌬tpa1⌬ double mutant would show increased sensitivity to MMS.…”

“…We also included a strain deficient in Stp22 in the study as a positive control. It has been reported that stp22⌬ cells are sensitive to these antibiotics because it is a component of the endosomal sorting complexes required for transport (ESCRT) complex that is involved in ubiquitin-dependent sorting of proteins into the endosome (52). As shown in Fig.…”