Abstract 413: Aneuploid cells depend on the RAF/MEK/ERK pathway and on RNA degradation for overcoming DNA damage and transcriptional burden

Abstract Aneuploidy, a state of karyotype imbalance, is a hallmark of cancer. Understanding the broad implications of aneuploidy at molecular, cellular, and physiological levels is crucial for effectively targeting aneuploid cancer cells. Aneuploidy often results in specific stress responses within cells, which must adapt to for survival and growth. Exploring these adaptive mechanisms to aneuploidy open avenues for targeted cancer therapies. Here, we induced aneuploidy in non-transformed RPE1-hTERT cells, generating multiple stable clones each exhibiting varying degrees of chromosomal imbalances. Employing a comprehensive genomic profiling strategy, we analyzed six isogenic clones using whole-exome and RNA sequencing. We uncovered a complex landscape, where each degree of aneuploidy brought its own set of changes and challenges. Additionally, we explored their cellular dependency landscapes, performing genome-wide CRISPR/Cas9 screenings along with extensive drug screens. As previously reported, we observed and confirmed increased DNA damage and p53 pathway activation in the aneuploid cells. We also found that the aneuploid clones triggered the DNA damage response (DDR), leading to an increased resistance to further DNA damage. Interestingly, we observed that aneuploid cells showed increased activity in the RAF/MEK/ERK pathway. Moreover, these cells demonstrated a heightened sensitivity to a range of clinically significant drugs that target this pathway, in particular in response to both genetic and chemical inhibition of CRAF. The activity of CRAF was found to be functionally connected to the resistance against DNA damage induction, and the inhibition of CRAF making aneuploid cells more susceptible to chemotherapies that induce DNA damage. Next, we observed increased of RNA synthesis and degradation in the aneuploid cells, along with heightened activity in both the nonsense-mediated decay (NMD) and the microRNA-mediated mRNA silencing processes. As a result, aneuploid cells displayed increased sensitivity to the perturbation of several key components of RNA degradation pathways. Furthermore, the dependencies of aneuploid cells on RAF/MEK/ERK activity and on intact RNA degradation were confirmed through examining a broad range of human cancer cell lines, emphasizing their significance in human cancer. Altogether, our results provide a comprehensive resource of karyotypically stable cells representing a range of aneuploidy states, and they reveal previously unidentified weaknesses in aneuploid cells that may be crucial for therapeutic developments. Citation Format: Marica Rosaria Ippolito, Johanna Zerbib Zerbib, Yonatan Eliezer, Eli Reuveni, Sonia Viganò, Giuseppina De Feudis, Anouk Savir Kadmon, Alice Ratti, Sara Martin, Kathrin Laue, Yael Cohen-Sharir, Simone Scorzoni, Francisca Vazquez, Uri Ben-David, Stefano Santaguida. Aneuploid cells depend on the RAF/MEK/ERK pathway and on RNA degradation for overcoming DNA damage and transcriptional burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 413.