Association of encapsulation of CDK12 inhibitor with anti-tumour activity in colon cancer cell lines.

person Pedro Pérez-Segura Medical Oncology Department, Hospital Universitario Clínico San Carlos, Madrid, Spain info_outline Pedro Pérez-Segura, Cristina Diaz-Tejeiro, Alberto Ocana, Adrián Sanvicente, María Arenas-Moreira, Carlos Alonso Moreno, Cristina Nieto Jiménez

Authors person Pedro Pérez-Segura Medical Oncology Department, Hospital Universitario Clínico San Carlos, Madrid, Spain info_outline Pedro Pérez-Segura, Cristina Diaz-Tejeiro, Alberto Ocana, Adrián Sanvicente, María Arenas-Moreira, Carlos Alonso Moreno, Cristina Nieto Jiménez Organizations Medical Oncology Department, Hospital Universitario Clínico San Carlos, Madrid, Spain, Hospital Clínico San Carlos, Madrid, Spain, Hospital Clínico San Carlos and IdISSC, Madrid, Madrid, Spain, Instituto de Investigación Sanitaria Hospital Clínico San Carlos, Madrid, Spain, Centro Regional de Investigaciones Biomédicas, Universidad de Castilla La-Mancha, Albacete, Spain Abstract Disclosures Research Funding Other Foundation Instituto de Salud Carlos III and Cris cancer Foundation Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Unfortunately, despite the incorporation of new therapeutic options, the metastatic setting is still an incurable disease. Cyclin-dependent kinases (CDKs) are a large family of proteins involved in cell cycle and transcriptional regulation, and are highly expressed in colon cancer, such as CDK12. Here, we explore the antitumor activity of a novel CDK12 inhibitor (CDK12-IN-E9) in preclinical models of colon cancer, and its liposomal formulation (LP-CDK12-IN-E9). Methods: Expression of CDK12 at a genomic level was assessed using web tools such as the Colonomics database. CDK12-IN-E9 was encapsulated in a liposomal formulation to evaluate differences between the free and the liposome form. The effect of compounds on cell viability was assessed with MTT assays. Matrigel assays were performed to study invasiveness and wound assays for migration. In addition, cell cycle and apoptosis studies were carried out by flow cytometry with 7-AAD, annexin V staining, and western-blot for specific protein expression. DNA damage and caspase degradation assays were performed by immunofluorescence. Results: Genomic mapping of CDK12 showed a higher expression in CRC than in normal tissue ( p−value = 1.424e−43 ). The CDK12 inhibitor was formulated in liposomes LP-CDK12-IN-E9 with the following characteristics: a polydispersity index of 0.11 ±0.02, a hydrodynamic radius of 62.98 ±10.29 nm and a zeta potential of -27.12±1.65 mV. CDK12-IN-E9 induced an antiproliferative effect in SW620, SW480 and HCT116 colon cancer cell lines. LP-CDK12-IN-E9 displayed an EC50 two times higher compared to CDK12-IN-E9 and a higher reduction of invasiveness between free inhibitor and its liposomal formulation mainly in HCT116 ( p-value < 0.001 ). Cell cycle studies showed no significant differences between CDK12-IN-E9 and LP-CDK12-IN-E9, but there was a profound induction of cell death at the highest doses through apoptosis mediated largely by caspases. In parallel to the effect on cell death, an increase in histone H2AX phosphorylation and an increase in cleaved caspase-3 was observed, which also implies a large increase in DNA damage in tumor cells. In vivo studies are ongoing. Conclusions: In summary, LP- CDK12-IN-E9 showed more antitumoral activity than its free formulation in CRC cell lines mainly through a caspase-dependent induction of apoptosis. This data suggests that liposomal formulation of novel chemical entities can augment its antitumoral properties.

Pre-clinical