Abstract 897: Targeting ALDH1A1 and stemness regulatory networks in ovarian cancer

Abstract

Abstract High grade serous ovarian cancer (HGSOC) is the leading cause of death from gynecologic malignancies. Ovarian cancer stem cells (OCSCs) are hypothesized to contribute to the emergence of chemoresistant tumors. Aldehyde dehydrogenase 1A1 (ALDH1A1) is an intracellular enzyme that oxidizes toxic aldehydes to carboxylic acids and plays a role in controlling cell differentiation pathways. We have previously used ALDH1A1 activity as a functional marker to identify OCSCs and demonstrated that OCSCs persist after chemotherapy and drive recurrent, drug resistant disease. However, the mechanism by which ALDH1A1 maintains stemness phenotype remains poorly understood. To examine the effect of ALDH1A1 on cellular survival signals in OCSCs, we generated a novel ALDH1A1-specific small molecule inhibitor named compound 974. Treatment of HGSOC cell lines with compound 974 reduced ALDH enzyme activity (p<0.01) and inhibited stem-like properties including spheroid formation (p<0.01) and clonogenic survival (p<0.05). Compound 974 treatment inhibited (p<0.01) stemness genes Prom1, Bmi1, Oct4 and Nanog. ALDH1A1 inhibition also reduced (p<0.05) cisplatin IC50 and synergized (p<0.05) with cisplatin treatment. To further examine the effect of compound 974 to inhibit ALDH1A1 and consequently tumor initiation, mice were injected with 106, 105 and 104 OVCAR3 cells treated in vitro with compound 974 (5µM for 48h). In a parallel study, mice were injected with 106, 105 and 104 of ALDH1A1 knockdown or scrambled control cells. In both cases, tumor initiation was significantly delayed compared to the control, and extreme limiting dilution analysis further revealed that compound 974 reduced (p<0.05) CSC frequency compared to control. To better understand the mechanism by which ALDH1A1 maintains OC stemness, OVCAR3 cells treated with vehicle or compound 974 were analyzed by RNA-sequencing and bioinformatics. Transcriptomic analysis revealed that compound 974 significantly (FDR < 0.05, fold change > 2) downregulated expression of markers strongly associated with CSC phenotypes (NF-κB, CD44, Klf4, Sox9, Fzd7) and chemoresistance (ABCB1). Ingenuity Pathway Analysis for downstream regulators of differentially expressed genes revealed that the senescence pathway was significantly altered. Analysis by qRT-PCR revealed that compound 974 inhibited (p<0.01) chemotherapy-induced expression of senescence marker p21 and senescence associated secretory phenotype (SASP) genes including IL6, IL8, CXCL1 and CXCL3. Compound 974 also inhibited(p<0.05) the chemotherapy-induced beta-galactosidase activity. Collectively, our findings further support the functional role for ALDH1A1 in OCSC and reveal a novel role for the enzyme in chemotherapy-induced senescence and disease relapse. Citation Format: Vaishnavi Muralikrishnan, Fang Fang, Tyler C. Given, Ram Podicheti, Douglas B. Rusch, Thomas D. Hurley, Kenneth P. Nephew. Targeting ALDH1A1 and stemness regulatory networks in ovarian cancer [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 897.