Abstract 2863: CDKN1A prevents IR-induced apoptosis in rhabdomyosarcoma tumor via repressing the pro-apoptotic factor NOXA in a novel apoptotic regulatory axis

Abstract

Abstract Rhabdomyosarcoma (RMS) is a tumor of the muscle and is the most common soft tissue cancer in children and teens, with approximately 400 to 500 new cases every year in the United States. While we have a good understanding of the interplay between pro- and anti-apoptotic regulators, much remains to be learned about how the decision to trigger apoptosis or not is controlled at the transcriptional level in RMS tumors. We strive to elucidate how pathways that drive apoptosis avoidance are epi-genetically pre-programmed in cells. We have discovered that SNAI2, a transcriptional repressor, acts as a master switch to either activate or dampen apoptosis in response to ionizing radiation (IR). While elevated SNAI2 results in transcriptional repression of the pro-apoptotic factor BIM and protection from apoptosis post-IR, a small number of SNAI2 null cells survive radiation to reproliferate in vivo, revealing the existence of hitherto unappreciated mechanisms that support the survival of irradiated cells. Hence, we hypothesized that there are additional genes or pathways in cells lacking SNAI2 that are co-opted to drive relapse disease. We have identified that CDKN1A or p21 expression is robustly increased in SNAI2 null cells. CDKN1A is thought to be a tumor suppressor. However, our preliminary data indicate that CDKN1A is an oncogene, and in RMS patients, high tumor expression is associated with poor outcomes. We found that CDKN1A expression in combination with SNAI2 can predict resistance to radiation across RMS cell lines. We next developed control RD, Rh30, and Rh18 cells, either CDKN1A or both CDKN1A and SNAI2 ablated using CRISPR/Cas9 reagents with control cells expressing control gRNAs. Ablation of CDKN1A, while not affecting proliferation, led to a significant increase in apoptosis post-radiation, and this effect was increased in SNAI2/CDKN1A double knockout cells. Further, in colony-forming assays, we find that double-ablated cells form significantly fewer colonies than control and single knockout cells. Loss of CDKN1A, SNAI2, or CDKN1A/SNAI2 led to a G2/M cell cycle block, indicating that the cell cycle effects of p21 might not be responsible for the phenotypes observed. Ongoing xenograft experiments have determined the effect we observe in vitro will also translate in vivo. Given that SNAI2 and CDKN1A are heterogeneously expressed, respectively, we will interrogate CDKN1A for its role in protecting differentiated cells from apoptosis. We will determine if CDKN1A prevents apoptosis by repressing the pro-apoptotic factor NOXA in a novel apoptotic regulatory axis specific to differentiated cells. Our study will show that critical primary and alternative apoptosis resistance mechanisms are epi-genetically pre-programmed in cells by SNAI2. Citation Format: Paulomi S. Modi, Myron Ignatius, Long Wang, Prethish Sreenivas. CDKN1A prevents IR-induced apoptosis in rhabdomyosarcoma tumor via repressing the pro-apoptotic factor NOXA in a novel apoptotic regulatory axis [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 2863.