Abstract 5987: Differential targets engaged by narazaciclib in comparison to the approved CDK4/6 inhibitors contribute to enhanced inhibition of tumor cell growth
Authors: Kechagioglou Petros , Dupont Camille , Yurugi Hajime , Distler Ute , Tenzer Stefan , Chernobrovkin Alexey , Riegel Kristina , Mooz Juliane , Lambert Mahil , Dötsch Volker , Cosenza Stephen , Fruchtman Steven M. , Rajalingam Krishnaraj 
Published: April 2023

Abstract
CDK4/6 inhibitors are clinically approved for the treatment of HR+, HER2- metastatic breast cancer by reinstating the G1/S checkpoint in tumor cells. Despite clinical benefit, safety concerns such as neutropenia and diarrhea, and disease progression, raises a critical need to identify novel therapeutic strategies. Narazaciclib (ON123300), novel CDK4/6i, designed to enhance efficacy and safety by its multi-targeted kinase inhibitor activity at low nM concentrations against CDK4/6, ARK5, CSF1R, and c-Kit. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; studying different administration regimens. Since several oncogenic signaling pathways are affected, we examined the efficacy of ON123300 in various breast cancer cell lines, and consolidate its mechanism of action by identifying the other targets engaged by ON123300. To identify direct and secondary targets engaged by narazaciclib and palbociclib, proteome wide Cellular Thermal Shift Assay (CETSA) was performed. To investigate narazaciclib’s effect on signaling pathways, Integrative Inferred Kinase Activity (INKA) was performed. Bioinformatics analysis explored the potential clinical effect of the identified targets. Molecular docking simulations investigated the potential interactions of narazaciclib to engage the new targets compared to other CDK4/6i. Both CETSA and INKA analysis revealed more potential targets engaged by narazaciclib compared to palbociclib in both MDA-MB-231 lysates and intact cells such as BUB1, CHEK1, AURKA, GSK3α and GSK3β. In TNBC patients with BUB1 overexpression bioinformatics analysis indicates a low survival correlation. Docking data showed a higher affinity of narazaciclib with BUB1 compared to palbociclib and abemaciclib. A stronger induction of apoptosis and senescence was detected in narazaciclib treated MMTV-PYMT cells, a murine mammary carcinoma model, compared to the other CDK4/6i, while narazaciclib enhanced CCL5 and CXCL10 mRNA levels. Reduction of PD-L1 protein levels and a promoting effect on the H2D1 and B2M mRNA levels was perceived in narazaciclib treated PYMT cells. Lastly, inhibition of autophagy, both at the early and late stages, may sensitize cancer cells to narazaciclib and induce irreversible cell proliferation inhibition, providing a novel therapeutic approach. Our data unveiled the differential targets engaged by narazaciclib in comparison to palbociblib. Narazaciclib treatment led to BUB1 protein degradation, overexpression of which is associated with poor prognosis in TNBC. Combination of narazaciclib with autophagy inhibitors sensitized several breast cancer cells to cell death. Narazaciclib treatment may promote antitumor immunity by influencing the expression of various immune modulators in the tumor cells which needs to be further validated in preclinical animal models; and ultimately in the clinic.
Citation Format: Petros Kechagioglou, Camille Dupont, Hajime Yurugi, Ute Distler, Stefan Tenzer, Alexey Chernobrovkin, Kristina Riegel, Juliane Mooz, Mahil Lambert, Volker Dötsch, Stephen Cosenza, Steven M. Fruchtman, Krishnaraj Rajalingam. Differential targets engaged by narazaciclib in comparison to the approved CDK4/6 inhibitors contribute to enhanced inhibition of tumor cell growth. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5987.