Novel multitarget small molecule inhibitor LCI133 and synthetic lethality in endometrial carcinoma via CDK9/PI3K axis.
Authors: Delara Ritchie , Pal Dhananjaya , Maddeboina Krishnaiah , Naumann R. Wendel , Crane Erin , Brown Jubilee , Mangum Arditti Page , Dryden Hailey L. , Yada Bharath , Sha Wei , Durden Donald , McHale Cody 
Published: June 2023

e15118 Background: Despite advances in molecular characterization of endometrial carcinoma (EC), options for effective targeted therapy against EC remain limited. PI3K/PTEN/AKT/mTOR signaling is the most frequently mutated pathway in EC, but targeted inhibition often results in resistance via compensatory signaling. In response, the Molecular Targeted Therapeutics Laboratory has designed multidimensional small molecules in silico to (1) target compensatory molecular signaling pathways to induce synthetic lethality, (2) circumvent combination therapy-induced toxicity, complicated pharmacokinetics, and undesirable drug-drug interactions by placing each inhibitory domain in a single target inhibitor, and (3) prevent acquired resistance by inhibiting kinome adaptation. The objective of this study is to assess the synthetic lethality response generated in EC by combinations of PI3K, BRD4, CDK4/6, and CDK9 inhibition through multitargeted small molecule inhibitors LCI132, LCI133, and LCI136. Methods: Following primary screening in a cell-free assays for potency against target proteins, LCI132, LCI133, and LCI136 were screened against five EC cell lines for 48 hours; and cell viability assays were performed. Two EC cell lines (AN3 CA and RL95-2) were chosen for subsequent mechanistic studies and were treated with multitarget or single agent inhibitor for 24 hours; and cells were assessed by flow cytometry for cytostatic or cytotoxic response, and lysates were prepared accordingly for Western blot (WB) analysis. EC cells were also treated as indicated with multitarget or single target inhibitors for time-course assessment of MYC, HEXIM1, and MCL1 relative gene expression by qRT-PCR. Results: EC cell lines showed differential sensitivity to multitarget inhibitors with LCI133 proving most broadly potent, particularly against AN3 CA (IC50 248 nM) and RL95-2 (105 nM). LCI133 induced significant G2 phase cell cycle arrest in AN3 CA ( pFDR< 0.05) and the apoptotic response in AN3 CA ( pFDR< 0.05)and RL95-2 ( pFDR< 0.05). qRT-PCR revealed all LCI compounds decreased AN3 CA HEXIM1 and MCL1 gene expression in a time-dependent manner, with LCI133 inhibiting expression up to 24 hours post-treatment. In AN3 CA, LCI133 dose-dependently decreased phosphorylation of RNA polymerase II (S2) and Akt (S473) and decreased total MCL-1 protein levels as assessed by WB, suggesting on-target simultaneous inhibition. Further studies revealed that LCI133 induces apoptosis in a caspase-dependent manner, resulting in increased levels of cleaved PARP protein. Conclusions: Multitarget PI3K/CDK4/6/9 inhibitors LCI133 and LCI136 are nanomolar potent in AN3 CA and RL95-2 EC cell lines. LCI133 ablated expression of antiapoptotic protein MCL-1 and induced caspase-dependent apoptosis in AN3 CA, implicating PI3K and CDK9 inhibition as a synthetic lethality combination in PTEN/PI3K co-mutated EC.