Abstract 372: Loss of <i>RB1</i> sensitizes <i>TP53</i>-mutated cancer cells to WEE1 inhibition by azenosertib

Abstract The retinoblastoma protein 1 (RB1), encoded by RB1 gene, plays a critical role in regulating the G1 to S-phase transition of the cell cycle through its interaction with the E2F family of transcription factors. In addition, RB1 has been shown to directly regulate DNA damage response and apoptosis. Loss of RB1 function, predominately via gene deletion, occurs frequently in various human cancers, with the highest prevalence in small cell lung cancer (almost ubiquitous) and basal-like breast cancer (up to 72%). RB1 loss is often associated with aggressive phenotypes, especially in cancers that harbor concurrent TP53 mutations. In TP53 mutated cancer cells, further loss of RB1 can drive uncontrolled entry into S phase, leading to increased replication stress and DNA damage. WEE1 kinase regulates essential cell cycle checkpoints, preventing premature mitosis and allowing time for DNA repair. Therefore, we hypothesized that loss of RB1 would sensitize TP53-mutant cancer cells to the selective WEE1 inhibitor azenosertib. We tested this hypothesis using small cell lung cancer (SCLC) and triple-negative breast cancer (TNBC) models. In TP53-mutant SCLC (N=9) and TNBC (N=3) cell lines, concurrent RB1 loss increased sensitivity to azenosertib, as evidenced by greater cell growth inhibition compared to cells with wild-type RB1 (median GRmax = -0.23 vs -0.81, 0.01 vs -0.55, respectively). Knockdown of RB1 in RB1 wild-type DMS53 (SCLC) and MDA-MB-231 (TNBC) cell lines sensitized them to azenosertib (GRmax = -0.47 vs -0.94, -0.02 vs -0.5, respectively). Downstream signaling analysis revealed that azenosertib treatment induced higher levels of DNA damage and apoptosis, as shown by the significant increase in γH2AX and cleaved-Caspase 3 levels in RB1 knockdown cells compared to control cells. Conversely, overexpression of RB1 in RB1-null cell lines significantly desensitized the cells to azenosertib with GRmax increased from -0.81 to -0.45 in NCI-H1048 (SCLC), and from -0.63 to -0.34 in MDA-MB-468 (TNBC), respectively. Consistently, RB1 overexpressed cells demonstrated lower levels of DNA damage and apoptosis after azenosertib treatment. In xenograft models, azenosertib demonstrated enhanced tumor growth inhibition (TGI) in models with both TP53 and RB1 loss (NCI-H146 [SCLC]: 90%, MDA-MB-468 [TNBC], 87%) compared to those with TP53 mutation alone (DMS53 [SCLC]: 49%, MDA-MB-231 [TNBC] 50%). Together, our data demonstrates loss of RB1 sensitizes TP53-mutant cancer cells to WEE1 inhibition in multiple models of SCLC and TNBC, suggesting RB1 loss can be used as a predictive marker to identify TP53-mutated patients of these cancers who are likely to benefit from WEE1 inhibition by azenosertib. Citation Format: Daehwan Kim, Olivier Harismendy, Catherine Lee, Erika Cabrera, Danielle Jandial, Doris Kim, Mark R. Lackner, Jianhui Ma. Loss of RB1 sensitizes TP53-mutated cancer cells to WEE1 inhibition by azenosertib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 372.