Abstract 1900: PTEN loss of function encodes sensitivity to multitargeted small molecule CDK4/6-CDK9-AURKA/B inhibitor, LCI133, and other CDK9 inhibitors.

Abstract ObjectiveTargeted therapies for endometrial cancer (EC) have shown promise, making multitargeted therapeutics of particular interest. LCI133 is a novel, in silico-designed triple inhibitor of CDK4/6-CDK9-AURKA/B that demonstrated nanomolar potency by 48-hour IC50 and selective dose-dependent apoptosis induction in PTEN mutant EC cell lines. In vivo, LCI133 showed antitumor activity against PTEN mutant EC xenografts and presented an acceptable safety profile. Given these data, our objective is to define the mechanism by which PTEN status serves as a biomarker for LCI133 and other transcriptional inhibitor sensitivity in EC. Methods1. PTEN WT HEC-1-B cells were transfected with PTEN shRNA and control shRNA to generate PTEN knockdown and control HEC-1-B cell lines. PTEN null AN3 CA cells were transduced with lentivirus to generate a doxycycline (Dox) inducible PTEN WT AN3 CA cell line.2. Isogenic in vitro studies were performed to determine potency of LCI133 and AZD4375 (single agent CDK9 inhibitor) by luminescent viability assay, 7-AAD/Annexin V and DNA dye flow cytometry assays. Nascent RNA (nRNA) was detected by Click-it Nascent RNA Capture Kit and assessed by qRT-PCR or confocal microscopy. Co-immunoprecipitation (Co-IP), reciprocal Co-IP, and Western blotting were used to examine protein-protein interactions.3. HEC-1-B PTEN shRNA or control shRNA xenografts were established in NSG mice, which were randomized and treated with LCI133 (50mg/kg) or vehicle (VEH) by daily intraperitoneal injection for 21 days. Tumor volume and animal mass were monitored, and biosamples were collected at end point. Results1. PTEN knockdown in HEC-1-B cells increased sensitivity to LCI133 and AZD4375 compared to control by viability assay (p ≤ 0.0001) and apoptosis assays (p ≤ 0.001). Conversely, Dox induced PTEN expression reduced sensitivity to LCI133 by apoptosis assay (p ≤ 0.001).2. In isogenic HEC-1-B cells, PTEN knockdown resulted in augmented relative nRNA transcription, followed by a significant decrease in nRNA after LCI133 treatment. A PTEN-dependent protein-protein interaction between RNA Polymerase (Pol) II and PTEN was shown in isogenic HEC-1-B-shRNA and PTEN-reconstituted AN3 CA cells.3. Tumors reached 50-75 mm3 more quickly in the HEC-1-B PTEN-shRNA group compared to control (p ≤ 0.0001). In the PTEN knockdown group, treatment with LCI133 resulted in slower tumor growth compared to VEH (p ≤ 0.0001), while in the control-shRNA group, there was no difference in tumor growth between LCI133 and VEH. ConclusionsThese data prove that the absence of PTEN is causally linked to sensitivity to LCI133 and AZD4375 via facilitating RNA Pol II dependent transcriptional elongation followed by a marked decrease in nRNA in PTEN knockdown EC cells. PTEN expression may be useful as a biomarker in selecting targeted therapeutics for EC driven through transcriptional activation. Citation Format: Halle Goodwin, Qi Zhang, Vaidehi Mujumdar, Krishaniah Maddeboina, Cody McHale, Bharath Yada, Hailey Dryden, Robert Wendel Naumann, Yovanni Casablanca, Erin K. Crane, Jubilee Brown, Allison Puechl, Brittany Lees, Donald Durden, Dhananjaya Pal. PTEN loss of function encodes sensitivity to multitargeted small molecule CDK4/6-CDK9-AURKA/B inhibitor, LCI133, and other CDK9 inhibitors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1900.