(T0930-03-18) Targeting Multiple Oncogenic Drivers in NSCLC: Therapeutic Potential of ARV-825 and Gefitinib-Based PROTAC-3 Protein Degraders

Purpose: Lung cancer ranks among the most prevalent malignancies worldwide. Current therapeutic options for lung cancer primarily involve EGFR inhibitors like erlotinib and gefitinib. However, current treatments present challenges of severe side effects, drug resistance, and financial burdens, often leading to patient noncompliance. We propose a novel synergistic formulation causing simultaneous degradation of EGFR and BRD4 to suppress c-MYC, EGFR, and K-RAS pathways, offering a potential therapy for NSCLC with diverse genetic profiles (Fig1A). Proteolysis Targeting Chimeras (PROTAC) emerges as a promising tool, targeting protein degradation over mere inhibition, thus representing an innovative approach to effectively reduce protein levels in lung cancer therapy. We aim to investigate the combination of BRD4 degrader (ARV-825/BPRO) and EGFR degrader (Gefitinib-based PROTAC-3/EPRO), referred to as BE-PRO. The objectives of our study are: (a)To investigate synergism utilizing BE-PRO in EGFR wild-type cells (H460), EGFR TKI sensitive cells (PC9), and EGFR TKI resistant cells (H4006 gefitinib resistance, H4006/GR) cells. (b) To investigate anticancer activity of BPRO, EPRO and BE-PRO in 2D and 3D models of NSCLC. (c) Optimization of BPRO and EPRO loaded depot formulation (BERD).

Methods: The IC₅₀ of BPRO and EPRO and the combination index (CI) of BE-PRO were determined by MTT assay on diverse NSCLC cell lines. Additionally, a cell viability study on HEK-293 cells was performed to test its selectivity against cancerous cells. The ability of single cells to survive the treatment and grow in colonies was investigated using a clonogenic assay in H460 and PC9 cells. A multicellular 3D spheroid model of H460 cells was developed to mimic in vivo tumor conditions and treated with all three groups for acute and chronic treatment regimens. A cell Titer-Glo® 3D assay and in vitro Live/Dead cell assay were performed to assess 3D cell viability and the apoptotic effect of the treatment groups on 3D spheroids of H460 cells, respectively. For the formulation optimization, Float-A-Lyzers were employed to assess the drug release from depot formulations and compared with tube method (Fig 3A,3B, and 3C). To evaluate precipitation in BERD, 50 µl of depot formulation was injected into the release media and observed under the stereomicroscope at different time points. To simulate subcutaneous drug release in vitro, agarose gel-based phantoms were cast as tissue mimics in Float-A-Lyzers, and the release pattern was optimized by injecting a depot loaded with a fluorescent dye to investigate the release pattern. The Float-A-lyzers were imaged under the IVIS Lumina LT series III instrument at different time points. Phantoms prepared with or without 0.5%w/v Lecithin were used to evaluate the efficiency of BPRO Depot against BPRO solution over a duration of 96h. To optimize the drug-to-polymer ratio in depot formulations, three different groups were evaluated for drug release by loading 10%w/v BPRO in varying concentrations of co-polymer (20%w/v, 25%w/v, 30%w/v).

Results: BE-PRO revealed strong synergism at molar ratio of 1:1 with a CI of 0.25±0.026 in H460 and 0.151±0.047 in PC9 cells (Fig 1B) while significantly inhibiting the clonogenic activity with 200 nM in H460 and PC9 confirming its antiproliferative and metastatic activity (Fig1D,1E and 1F). Over 85% cell viability was observed with treatment of BPRO, EPRO and BE-PRO in HEK-293 cells (Fig1C) as HEK-293 has low EGFR and c-MYC expression, which suggest that it is selective towards lung cancer cells not healthy cells. Approximately 74.50±2.14% and 53.33±3.87% inhibition in the growth of H460 3D spheroids was observed on day 9 for chronic and acute exposure with BE-PRO. The acute treatment regimen showed a non-significant decrease in cell growth compared to chronic treatment exposure (Fig2A,2B and 2C). The BE-PRO group resulted in 55.63±1.24% cell viability when treated for 6h whereas similar concentration reduced the cell viability to 28.31±3.458% when exposed to multiple doses compared to control (Fig2D and 2E). From the in vitro Live/Dead Cell assay, higher red fluorescence was found in treatment groups as compared to control confirming large number of apoptotic cells in both acute and chronic exposure (Fig2F and 2G). No precipitation was observed in depot formulations until 4h (Fig3D). BPRO solution shows >75% dissolution within 6h, whereas the BPRO depot containing PCL-PEG-PCL (25%w/v) and NMP as the solvent showed less than 10% drug release in 6h (Fig3G). The effect of varying concentrations of BPRO and co-polymer on the in vitro release profile is summarized in (Fig3F). Fluorescent IVIS images of tissue-mimicking phantoms demonstrating sustained release of dye-loaded depot formulations (Fig3E).

Conclusion: The selective degradation of BRD4 and EGFR, targeting the three major oncogenic drivers in NSCLC, presents an exciting therapeutic approach for KRAS and EGFR mutant lung adenocarcinoma. The IVIS images distinctively show that our tissue-mimicking phantom effectively withholds the dye-containing depot formulation for sustained release, unlike media-filled tubes that exhibit complete release of dye and fail to withhold the depot. The newly developed depot formulation, designed for sustained release of dual PROTACs, is expected to enhance efficacy at a reduced dosage.

Acknowledgements: This work was funded by the National Institutes of Health (NIH) grant R15 (1R15CA271133-01A1)