Abstract Mutated KRAS and TP53 are well recognized drivers of multiple human cancers including lung cancer, the leading cause of cancer-associated deaths. Yet, directly targeting mutated KRAS or restoring wildtype p53 levels in KRAS/p53-mutated tumors still remains a therapeutic challenge. A rational alternative approach involves targeting potentiators of KRAS;p53-driven tumorigenesis, which represents one of the research ambitions pioneered by the National Cancer Institute (NCI) RAS initiative to meet this challenge. Therefore, we aimed to identify genes, that when lost, potentiate cellular transformation of a non-transformed KRAS;TP53-mutated human bronchial epithelial cell line (HBEC-KP). To address this question, a genome-wide selection experiment was performed using CRISPR/Cas9. Anchorage-independent (AI) growth, a hallmark of cancer, was selected as a phenotypic readout for one experiment. Without additional genetic perturbation, wildtype HBEC-KP cells were incapable of AI growth. Only certain mutants were capable of supporting colony formation in soft agar. These clones were isolated and the integrated small guide RNAs (sgRNAs) were identified. Genes that are targeted by the sgRNAs were then individually knocked out for validation. Through this approach, we discovered that loss of ARPC3 is sufficient for promoting AI growth in the HBEC-KP cells. This contribution to AI growth is attributed to the role that ARPC3 plays in regulating Arp2/3 activity, as inactivating the Arp2/3 complex using a small molecule inhibitor, CK666 also promoted colony growth in the HBEC-KP cells. We determined that ARPC3 loss was not sufficient to drive AI – in the absence of KRAS activation and p53 loss colony growth was not observed. This indicates the essential role that mutated KRAS and/or p53 play to support AI. To test if mutated KRAS is essential for the phenotype, the Arp2/3 complex was inactivated in KRAS-mutated HBECs, which supported AI growth. This suggests that mutated KRAS and loss of Arp2/3 activity are sufficient for driving AI growth in HBECs. However, combined loss of p53 and ARPC3 failed to drive the phenotype, implicating that mutated KRAS is required. In addition, the Arp2/3 complex is essential for maintaining lamellipodial shape via actin filament network and cell motility, which was validated in our HBEC-KP-ARPC3 knockout cells. However, this protein complex is also known to promote AI growth and development of various cancers, as opposed to acting as a suppressor for growth. Multiple mechanisms that lead to this growth phenotype are currently being explored. Clinical relevance is also being closely examined to further understand the role of the Arp2/3 complex in cancer. Citation Format: Chennan Li, Andrea L. Kasinski. Loss of Arp2/3 activity promotes anchorage independent growth in KRAS;TP53-mutated human bronchial epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 838.