Abstract 3006: Efficacy of novel CDK9/CDK4/6/PI3K triple inhibitors, LCI133 and LCI139, in Ewing sarcoma

Abstract Introduction: The survival rate of patients with Ewing sarcoma (ES) has seen slow improvement over the past several decades and remains dismal for those with recurrent or metastatic disease. Initial clinical trials in relapsed/refractory ES with single agent targeted therapies, or combinations of single agents, have not yet improved patient outcomes. To overcome this barrier, the Molecular Targeted Therapeutics Laboratory has developed by in silico design “first in class” small molecule multikinase inhibitors (MKIs) which simultaneously target multiple tumor-promoting pathways with a single drug at nM potency. LCI133 (CDK4/6/CDK9) and LCI139 (PI3K/CDK4/6/CDK9) potently inhibit key kinases that control mitogenic signaling, proliferation, and transcription. The purpose of this study is to evaluate the potency, efficacy, and toxicity of LCI133 and LCI139 in ES model systems. Methods: ES cell lines and normal human skeletal muscle cells (SkMC) were treated with MKIs in logarithmic fashion and IC50s were determined by CellTiter-Glo 2.0 viability assays. Incucyte® Annexin V Dye was used to evaluate apoptosis over time by Incucyte® S3 following MKI treatment. Cell cycle analysis was performed over time using Incucyte® Cell Cycle Lentivirus Reagent and analyzed using an Incucyte® S3. Western blot and qPCR analysis was used to assess protein and RNA expression changes, respectively, of targeted pathways to confirm on-target activity of these MKIs following 3-24 hr treatment. Results: LCI133 and LCI139 were nanomolar potent in ES cell lines TC71 (392.5nM/312nM) and A673 (471.5nM/407.4nM), while only LCI139 was nanomolar potent in the RDES (262.2nM) cell line. Critically, LCI133 and LCI139 were less toxic against SkMC compared to single agent CDK9 inhibitors. In TC71 and A673 cells, both compounds induced statistically significant dose-dependent apoptosis, as well as G1 phase cell cycle arrest. Three hr LCI133 and LCI139 treatment decreased expression of CDK9-RNA polymerase II response genes MCL1 (P ≤ 0.001) and MYC (P ≤ 0.001). 24 hr treatment had differential effects on pRb and pAKT levels. pRpb1 and MCL-1 were reduced in all cell lines by LCI139 and/or LCI133. Increased cleaved PARP (cPARP) was observed in all cell lines following 24 hr treatment with LCI133 and LCI139 confirming the induction of apoptosis. Conclusion: Novel small molecule MKIs allow for the simultaneous targeting of multiple pathways while mitigating the toxicity of single agent combination therapy. This is a promising potential treatment strategy for ES patients for whom new treatment approaches are desperately needed. These studies provide preclinical evidence of the potential efficacy of small molecule MKIs in ES and support the future efforts of in vivo models evaluating efficacy and safety of these agents. Citation Format: Kaitlyn H. Smith, Cody C. McHale, Kenzie Wells, Krishnaiah Maddeboina, Kimberly Q. McKinney, Poornima Gourabathini, Jeffrey S. Huo, Donald L. Durden, Javier Oesterheld. Efficacy of novel CDK9/CDK4/6/PI3K triple inhibitors, LCI133 and LCI139, in Ewing sarcoma [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 3006.