Abstract SY14-03: Exploiting dysregulated ribosomal homeostasis in chromosome 9p21.3 deleted cancers and microsatellite unstable cancers

Synthetic lethality, a phenomenon in which alterations in two or more genes together induce cell death while changes in either gene alone do not, provides a promising approach for cancer treatment. Indeed, various inhibitors targeting synthetic lethal interactions are approved by the Food and Drug Administration or in clinical trials. Seeking to nominate additional synthetic lethal targets, we analyzed large-scale CRISPR knockout screening data and identified Pelota mRNA surveillance and ribosome rescue factor (PELO) as a promising therapeutic target for two independent and frequently observed molecular subtypes of cancer: biallelic deletion of chromosomal region 9p21.3 (9p21.3-/-) and microsatellite instability-high (MSI-H). Homozygous deletion of chromosomal region 9p21.3 is one of the most frequently observed somatic copy number alterations in human cancers, occurring in approximately 15% of all cancers. Many 9p21.3-/- cancers are associated with poor clinical outcomes including subsets of glioblastoma, mesothelioma, urothelial, pancreatic, esophageal, and non-small cell lung cancers. The primary driver of 9p21.3-/- is thought to be loss of the tumor suppressor cyclin-dependent kinase inhibitor 2A. Additionally, studies have linked the loss of the interferon gene cluster on 9p21.3 to immune evasion and primary resistance to immune checkpoint inhibitors. Extensive deletions of chromosomal regions can provide opportunities for cancer treatment. For instance, PRMT5 and MAT2A were found to be promising targets in 9p21.3-/- cancers due to synthetic lethal interactions with deletions of the 9p21.3 gene methylthioadenosine phosphorylase. While clinical trials are underway examining PRMT5 and MAT2A inhibitors, the frequency and poor outcomes of many 9p21.3−/− cancers underscores the urgent need for additional therapies to treat this diverse group of cancers. Using large-scale functional genomic datasets, we identified PELO as the top preferential dependency in 9p21.3-/- cell lines. We also observed that some cell lines with intact 9p21.3 (9p21.3+) were dependent on PELO for survival. Examining other genomic features, we found that these cells were characterized as MSI-H, a hypermutable state observed in subsets of colon, endometrial, gastric, and ovarian cancers. Indeed, when we compared 9p21.3+/MSI-H and 9p21.3+/microsatellite stable (MSS) cell lines, we found that PELO scored as a strong preferential dependency in MSI-H cell lines, second only to the previously described synthetic lethal target Werner helicase (WRN). To validate these findings, we interrogated the viability effects of PELO knockdown across a panel of cell lines representing 9p21.3+/MSS, 9p21.3-/-/MSS, or 9p21.3+/MSI-H. CRISPR interference (CRISPRi)-mediated PELO knockdown with three distinct guide RNAs impaired the viability of 9p21.3-/-/MSS cell lines and 9p21.3+/MSI-H cell lines, but not 9p21.3+/MSS cell lines. We also sought to validate PELO dependency in tumor organoid models for which pre-existing CRISPR data were unavailable, and therefore, we could only predict their dependence on PELO based on the two identified biomarkers. We demonstrated that 9p21.3-/- or MSI-H tumor organoids were preferentially dependent on PELO as compared to 9p21.3+/MSS models. We also sought to evaluate the effect of PELO knockdown in tumor maintenance in vivo. Leveraging a doxycycline-inducible CRISPRi system to suppress PELO expression, we showed that PELO knockdown significantly shrank 9p21.3-/- tumors grown on the flanks of nude mice. We next pursued the culprit lesion in 9p21.3-/- cancers that conferred PELO dependency. Hypothesizing that the loss of a 9p21.3 gene confers PELO dependency, we performed a focused loss-of-function CRISPR/Cas12a screen targeting 9p21.3 genes in the context of PELO knockdown or control conditions. We identified the mRNA surveillance gene FOCAD as a 9p21.3 gene whose knockout lead to preferential impairment in cells with PELO depletion. This led us to hypothesize that FOCAD deletions were conferring PELO dependency. In focused validation, we showed that FOCAD deletion sensitizes cell lines to PELO knockdown. Furthermore, we discovered that restoring FOCAD rescued 9p21.3-/- cells from PELO depletion. Together, these data demonstrated FOCAD loss was both necessary and sufficient for PELO dependency in the 9p21.3-/- context. Next, we sought to determine the mechanistic basis for PELO dependency in MSI-H cancers. We hypothesized that a mutation associated with MSI-H sensitizes cells to PELO loss. We observed that deletions in the splicing acceptor site at intron 29 of the superkiller complex (SKIc) member tetratricopeptide repeat domain 37 (TTC37) was the most correlated microsatellite mutation with PELO dependency. Thymidine deletions in this splicing acceptor site were predictive of decreased TTC37 protein levels in cell lines. These data led us to hypothesize that functional loss of TTC37 is the culprit lesion that renders MSI-H cancers dependent on PELO for survival. Evaluating this hypothesis, we found that TTC37 knockout sensitized cells to PELO depletion. We also showed that exogenous expression of TTC37, but not control, cDNA rescued the viability of MSI-H cell lines from PELO knockdown. Taken together, these data demonstrated that functional TTC37 impairment is sufficient and necessary for PELO dependency in the MSI-H setting and could serve as a refined predictive biomarker for PELO dependency. We next sought to investigate how FOCAD loss and TTC37 mutations converge to confer PELO dependency. TTC37 interacts with Ski2-like RNA helicase (SKIV2L) and WD Repeat Containing Protein 61 (WDR61) to form the SKIc, which promotes the 3’-5’ exosome degradation of mRNA from stalled ribosomes. We examined the Cancer Cell Line Encyclopedia proteomics dataset and demonstrated positive correlations between FOCAD, TTC37, and SKIV2L. Consistent with these datasets, we observed decreased TTC37 and SKIV2L protein levels in 9p21.3-/- cell lines and MSI-H cell lines. Based on these results, we asked if FOCAD was required and/or sufficient to maintain TTC37 and SKIV2L protein stability. We expressed FOCAD cDNA in 9p21.3-/- cells and observed increased TTC37 and SKIV2L protein levels. Furthermore, FOCAD knockout reduced SKIV2L and TTC37 protein levels in 9p21.3+/MSS cells. Together, these data support FOCAD as a critical regulator of SKIV2L and TTC37 stability. We hypothesized that FOCAD deletions and TTC37 mutations were capturing SKIc loss, thereby increasing reliance on PELO for survival. We reasoned that if functional loss of the SKIc was responsible for PELO dependency, SKIV2L deletion would also confer increased PELO dependency. We knocked SKIV2L out and observed that loss of SKIV2L sensitized cells to PELO loss. These data support our hypothesis that loss of SKIc function confers increased dependence on PELO for survival. To assess how SKIc-deficient cancer cells respond to PELO suppression, we performed gene expressing profiling. We found that SKIc-deficient cells had a robust transcriptional response with gene set enrichment analyses demonstrating upregulation of the unfolded protein response, a signaling network responding to aggregated unfolded or misfolded proteins. We validated these findings by demonstrating that PELO depletion preferentially upregulated C/ebp homologous protein (CHOP) expression and induced X-BOX binding protein (XPB1) splicing, markers of UPR activation, in the SKIc-deficient setting. Together, our observations reveal that MSI-H associated mutations and large 9p21.3 deletions involving TTC37 and FOCAD, respectively, independently impair the SKIc and confer a synthetic lethal relationship with PELO. Since MSI-H and large 9p21.3 deletions are frequently observed in patients, a PELO-based therapeutic could have broad implications for clinical oncology. Citation Format: Patricia Borck, Isabella Boyle, Kristina Jankovic, Nolan Bick, Kyla Foster, Anthony Lau, Lucy Parker-Burns, Daniel Lubicki, Ashir Borah, Nicholas Lofaso, Sohani Das Sharma, Riya V. Kishen, Joshua Dempster, Francisca Vazquez, Edmond M. Chan. Exploiting dysregulated ribosomal homeostasis in chromosome 9p21.3 deleted cancers and microsatellite unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited s); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2): nr SY14-03.