Abstract Background: Based on the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), a special subtype of breast cancer which expresses all three receptors can be distinguished, namely triple-positive breast cancer (TPBC). TPBC patients has a higher risk of recurrence and lower survival rate compared to other luminal breast cancers. However, there are few studies on predicting molecules of prognosis and treatment response for TPBC. Methods: Proliferation essential genes (PEGs) were identified using CRISPR-Cas9 dataset of DepMap. The expression profiles and clinical data of TPBC were obtained from the TCGA database, GEO datasets and our center cohort. To develop a TPBC-PEG prognostic signature, cox regression and Lasso (Least absolute shrinkage and selection operator) regression analysis were applied. Functional analysis was performed using Gene Set Enrichment analysis. Finally, the relationship between candidate genes and neoadjuvant therapy (NAT) sensitivity was explored using real-time qPCR (RT-qPCR) and immunohistochemistry (IHC) based on clinical samples. Results: Among 900 TPBC-PEGs, 437 genes showed significant differential expression between TPBC and normal tissues. Subsequently, we identified 3 prognostic PEGs (ACTL6A, CCT2, TARS) and used them to construct a PEG signature. Patients with high PEG signature scores exhibited worse overall survival and lower sensitivity to NAT compared to those with low PEG signature scores. RT-qPCR indicated that in patients who lacked sensitivity to NAT, ACTL6A and CCT2 were significantly upregulated. The IHC results showed that ACTL6A protein was highly expressed in the NAT-insensitive group and non-pathological complete response patients. Conclusion: In this study, we developed an efficient PEG prognostic model that can predict the outcome for TPBC. Furthermore, we found that the expression of ACTL6A is associated with neoadjuvant therapy sensitivity in TPBC patients and can serve as an important factor in predicting patient prognosis and drug sensitivity. Figure 3. Construction and validation of a PEG signature for TPBC patients. Figure 6. The predictive value of ACTL6A on NAT efficacy and its correlation with clinical characteristics. Citation Format: Xiaofen Li, Wenfen Fu, Shiping Luo, Jie Zhang, Qingshui Wang, Chuangui Song. Discovery of proliferation essential gene signature and ACTL6A as potential biomarker for predicting prognosis and neoadjuvant therapy response in triple-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-03.