Abstract 3157: Novel synthetic lethality-based cellular assays for cancer drug discovery

Abstract In the present study we illustrate how, using the concept of synthetic lethality (SL), we developed cellular assays suitable for the screening of new drugs targeting cancer-specific weaknesses. SL is a promising strategy in cancer therapy that exploits the genetic vulnerabilities of cancer cells. This approach targets pairs of genes where the mutation or changes of one gene does not affect cell viability, but the simultaneous occurrence of these events in both leads to cell death. This concept is particularly valuable in cancer treatment, as it allows for the selective targeting of cancer cells while sparing normal cells. In this study we coupled powerful molecular approaches such as CRISPR/Cas9 or RNA interference (siRNA and shRNA) with cell viability tests (CellTiter-Glo®), to generate cellular assays suitable for assessing SL of two different genes of interest (GOIs) in proof-of-concept studies and perform drug screening to identify novel inhibitors able to mimic loss or mutations of the GOIs, inducing specific cell death of tumor cells. First, we induced SL by silencing VPS4A in cells already exhibiting loss or reduced function of VPS4B, ultimately triggering selective tumor cell death while sparing normal cells. Indeed, the homozygous loss of VPS4B is seen in up to 4% of colorectal, ovarian, and pancreatic cancers. The SL was then assessed by using siRNA to silence VPS4A and VPS4B in various cancer cell lines expressing different levels of the 2 GOIs and evaluating cell viability. Conversely, to generate stable cellular assays, CRISPR/Cas9 technology was applied to develop both VPS4B knockout (KO) and heterozygous (HET) cell lines, validated through Sanger sequencing and protein expression analysis. These genetically modified lines were critical for understanding the molecular mechanisms driving SL, as they were further engineered to obtain an inducible VPS4A silencing (shRNA technology), allowing for controlled VPS4A depletion. These experiments demonstrated that VPS4A silencing in VPS4B KO cells significantly reduced cell viability, confirming the synthetic lethal relationship between the two GOIs. In conclusion, our survival assay approach could be applied to validate the SL between 2 GOIs, identify a suitable genetic context for applying customized therapeutic options, and new drugs to treat cancer. Proof of principle studies could be conducted in transient using siRNA to silence GOIs in various cancer cell lines expressing different levels of GOIs; while stable modulation of the expression of GOI might be obtained using ad hoc engineered cell lines, thus allowing long-term studies and screening campaigns. Cellular assays able to elucidate SL mechanisms may have a significant impact on the cancer drug discovery identifying therapies that specifically target cancer cells with known genetic alterations, while minimizing the impact on normal cells and therefore reducing potential side effects. Citation Format: Giovanna Maresca, Fiorella Scagnoli, Francesca Sassone, Alessandro Poli, Paola Bonetti, Laura Curti, Silvia Goldoni, Helena Batoulis, Ekaterina Nevedomskaya, Jean-Francois Rolland, Elisabeth Pook, Loredana Redaelli, Tom Grammatopoulos. Novel synthetic lethality-based cellular assays for cancer drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3157.