Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor characterized by rapid disease progression and poor patient survival. Key transcription factors implicated as drivers of unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Strategies to exploit these phenotypes for innovative precision medicine approaches will be impactful. Using an unbiased agnostic preclinical drug screen to uncover therapeutic opportunities in SCLC, we identified a strong signal with PLK1 inhibitors (PLK1i) with low nanomolar IC50. We extended the in vitro findings by testing the efficacy of PLK1i in vivo using traditional xenograft of SCLC H526 cell line and patient derived xenografts (PDX). Volasertib achieved significant tumor growth inhibition relative to control in H526 xenografts. Also, onvansertib significantly inhibited growth of platinum-resistant and platinum-sensitive PDXs. The combination of PLK1i with standard chemotherapeutic agents identified promising synergy of the combination of onvansertib and paclitaxel. We further interrogated for predictive biomarkers of PLK1i sensitivity using gene expression profile comparing highly sensitive to less sensitive cell lines. High expression of C-MYC but not PLK1, TP53 or RB was associated with resistance to PLK1i. Conversely, while TP53 expression level did not correlate, TP53 gene mutation status (inactivating disruptive mutations) correlated with cell sensitivity to PLK1i. We queried the publicly available CCLE and the Cancer Therapeutics Response Portal to evaluate whether any of the SCLC subtypes have therapeutic vulnerability to PLK1i. In general, high expression of YAP1 in SCLC cell lines correlated with greater sensitivity to PLK1i. A YAP1 positive cell line, SW1271, with strong TP53 expression was particularly resistant to PLK1i. CRISPR knockout of YAP1 in this cell line enhanced SW1271 sensitivity to PLK1i suggesting that YAP1 expression as a marker of vulnerability to PLK1i could be context dependent especially when co-occurring with TP53 mutations. The mechanism of this interaction will be discussed. Citation Format: John C. Schmitz, Guojing Zhang, Andrey A. Ivanov, Abbe Pannucci, Maya Ridinger, Taofeek K. Owonikoko. Targeting PLK1 effectively suppresses growth of small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4995.