Abstract PO5-24-01: Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models

Abstract

Abstract Despite improvement of outcomes for HR+, HER2- metastatic breast cancer by cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors, safety concerns and disease progression raise a critical need to identify novel approaches. Narazaciclib (ON123300), a novel CDK4/6i, designed to enhance efficacy and safety by its multi-targeted kinase inhibitor activity. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; studying different administration regimens and in endometrial cancer in combination with letrozole (NCT05705505). Our study explored the activity of narazaciclib and its metabolite ON1232580 in comparisons to the FDA approved CDK4/6i and identify additional targets engaged by narazaciclib. To identify direct and secondary targets engaged by narazaciclib and palbociclib, proteome wide Cellular Thermal Shift Assay (CETSA) and Integrative Inferred Kinase Activity (INKA) analysis were performed. Both CETSA and INKA analysis identified additional 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. Molecular docking data showed a higher affinity of narazaciclib with BUB1 compared to palbociclib and abemaciclib. The differential activity of narazaciclib was explored by using cohorts of human and mouse breast cancer cell lines and cell based assays. Comparison of narazaciclib’s in vitro IC50 profile to other CDK4/6i was studied against a panel of 370 kinases (HotSpot) and Kd values were determined by KINOMEscan. Intracellular IC50 kinase values were determined by NanoBret technology. Narazaciclib and abemaciclib were found to be the most promiscuous in vitro kinase inhibitors and ribociclib the most specific. Abemaciclib and narazaciclib had similar profiles against the CDK family members. Kd values of CDK4/cyclinD1 binding show similar trends; abemaciclib (0.08 nM), narazaciclib (0.18 nM), palbociclib (0.75 nM) and ribociclib (1.3 nM). Although narazaciclib displayed nM IC50 values in the in vitro assays against many CDKs, narazaciclib was very specific in cellular kinase assays with highest activity against CDK4/6, CSF1R and NUAK1. A stronger inhibition of cell proliferation, as well as induction of apoptosis and senescence were detected in narazaciclib and its metabolite ON1232580 treated PYMT cells, a murine mammary carcinoma model which recapitulates luminal BC subtype, compared to the other CDK4/6i. Both narazaciclib and ON1232580 enhanced CCL5 and CXCL10 mRNA levels, while a higher reduction of PD-L1 and pRb protein levels and a promoting effect on the H2D1 and B2M mRNA levels was shown in narazaciclib and its metabolite treated PYMT cells. Narazaciclib showed significant synergy with anti-PD1 in the EMT-6 syngeneic breast cancer model. A differential effect of narazaciclib on the expression levels of 62 cytokines was detected compared to abemaciclib and palbociclib. Lastly, inhibition of autophagy sensitizes cancer cells to both ON123300 and ON1232580, and induces irreversible cell proliferation inhibition, providing a novel therapeutic approach. Our findings identify important differences generated from assay models studying kinase inhibition, binding and pathway engagement. Understanding the role of the differential targets engaged by narazaciclib, the potential enhanced antitumor immunity and the sensitization by autophagy inhibitors to cell death, will guide future clinical studies. Citation Format: Petros Kechagioglou, Debomita Chakraborty, Camille Dupont, Hajime Yurugi, Ute Distler, Stefan Tenzer, Alexey Chernobrovkin, Kristina Riegel, Julianne Mooz, Mahil Lamber, Volker Dötsch, Stephen Cosenza, Steven Fruchtman, Krishnaraj Rajalingam. Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-01.