Abstract 1856: Synergistic combination of PROTACs degrading oncogenic proteins: In vitro studies and formulating a long-acting injectable for in vivo testing in lung tumor-bearing mice

Introduction: Lung cancer remains a leading cause of cancer-related mortality worldwide, with accountability of more than 85% of cases of non-small cell lung cancer (NSCLC). Current therapies, such as EGFR inhibitors (e.g., erlotinib, gefitinib), face significant challenges, including severe side effects, drug resistance, and financial burdens, leading to patient noncompliance. In recent years, Protein-Targeting Chimeras (PROTACs), a class of protein degraders, is a groundbreaking strategy that degrades, rather than merely inhibits, targets proteins. This work investigates a combination of PROTACs degrading key oncogenic proteins of lung cancer. BRD4 degrading PROTAC (BPRO) and an EGFR degrading PROTAC (EPRO) were combined to simultaneously degrade BRD4 and EGFR to suppress c-MYC, EGFR and K-RAS, offering therapeutic potential for NSCLC with diverse genetic profiles. Objectives: Our study objectives include (a) evaluating BE-PRO synergism in various NSCLC cell line i.e. wild-type EGFR (H460 cells), EGFR TKI-sensitive (PC9 cells), and EGFR TKI-resistant (H4006/GR) cells; (b) assessing anticancer efficacy of BE-PRO in 2D and 3D NSCLC models; and (c) optimizing a self-injectable biodegradable subcutaneous depot formulation (BERD) for sustained delivery; (d) to analyze the in vivo anticancer efficacy of BERD in NSCLC cells tumor xenograft model. Methods: In vitro assays - IC50 and combination index (CI), invasion assay, clonogenic assay, and 3D spheroids growth were carried out to identify the synergistic ratio of BPRO and EPRO in different NSCLC cell lines. 3D-cell viability and apoptosis were studied using Cell Titer-Glo® 3D and Live/Dead Cell imaging assays. Further, depot formulation was developed using a biodegradable polymer and tested using a subcutaneous tissue-mimicking phantom using IVIS imaging. To determine the in vivo efficacy of BERD, a subcutaneously engrafted HCC827 cells xenograft model has been developed, and currently, we are measuring tumor growth using ultrasound and vernier caliper methods. Results: The combination index and cytotoxicity interaction graph demonstrated of BPRO and EPRO (CI = 0.25±0.026 in H460; 0.151±0.047 in PC9, 0.693±0.013 in H4006/GR) and significant inhibition of invasion and clonogenic activity. The combination showed minimal toxicity in healthy cells, HEK 293, and lung fibroblast. In 3D spheroids, BE-PRO achieved ∼74.5% and ∼53.3% growth inhibition under chronic and acute dose regimens, respectively. BERD ensured sustained drug release over 72 hrs, with minimal precipitation and reduced initial burst effects compared to the individual drug solution. Conclusion: A sustained-release combination of BRD4 and EGFR degrading PROTACs could be a promising approach, tackling key oncogenic drives while minimizing dosing frequency and off-target toxicities. Citation Format: Akanksha Patel, Mukti Vats, Ketan Patel. Synergistic combination of PROTACs degrading oncogenic proteins: In vitro studies and formulating a long-acting injectable for in vivo testing in lung tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1856.