454 A novel MDK targeted therapy for the treatment of endometrial cancer

Objectives/Goals: Endometrial cancer (ECa) incidence is rising rapidly in women under 40 years of age, and response rates and progression-free survival remain poor in advanced ECa. New targeted therapies are urgently needed. The objective of this study is to develop and test a potent inhibitor that targets midkine (MDK) for treating ECa. Methods/Study Population: Using the three-dimensional structure of human MDK, we rationally designed a small organic molecule (HBS-101) that directly binds with MDK and that functions as a MDK inhibitor. Microscale thermophoresis assay (MST) and in silico docking studies were used to demonstrate direct binding of HBS-101 to MDK. In vitro activity was tested using MTT, clonogenicity, and apoptosis assays. Mechanistic studies were conducted using Western blot, RT-qPCR, STAT3 and NFkB reporter gene assays, RNseq, and TEM. Status of MDK in ECa was determined using TNMplot database. Pharmacokinetics, Maximum Tolerable Dose, and in vivo efficacy studies were conducted using mouse models. HBS-101 efficacy in chemotherapy-resistant ECa was demonstrated using therapy-resistant primary ECa (TR-ECa) cells generated in-house. Results/Anticipated Results: MDK is highly expressed in ECa tumors compared to normal tissues. MDK and its receptors are expressed in established and primary ECa cell lines. Knockdown of MDK reduced ECa cell proliferation and colony formation. HBS-101 directly binds to MDK with nanomolar affinity, and a potential interaction surface on MDK was identified. HBS-101 decreased ECa cell viability and colony formation with an IC₅₀ ranging from 0.5 to 5 µM. Mechanistic studies showed that HBS-101 suppressed STAT3 and NFκB activity and induced cell death, ER stress, and ferroptosis. TR-ECa cells exhibited sensitivity to HBS-101. HBS-101 possesses favorable drug-like properties, including oral bioavailability, in vivo stability and little in vivo toxicity up to 10 mg/kg. HBS-101 significantly inhibited tumor growth in ECa PDX models. Discussion/Significance of Impact: HBS-101 is a novel therapeutic agent that targets MDK via a unique mechanism, shows favorable pharmacokinetics, and induces apoptosis and ferroptosis in ECa cells by blocking MDK signaling. HBS-101 shows promise in treating high-grade and therapy-resistant ECa and may help overcome the current lack of good treatment options for these patients.