Combination of multivalent DR5 receptor clustering agonists and histone deacetylase inhibitors for treatment of colon cancer.

Death Receptor 5 (DR5) targeted therapies offer significant promise due to their pivotal role in mediating the extrinsic pathway of apoptosis. Despite DR5 overexpression in various malignancies and the potential of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), clinical applications of anti-DR5 monoclonal antibodies (mAbs) have been hampered by suboptimal outcomes potentially due to lack of receptor clustering. To address the limitation, we developed N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based conjugates integrating multiple copies of DR5-targeting peptide (cyclic WDCLDNRIGRRQCVKL; cDR5) to enhance receptor clustering and apoptosis. Three conjugates with variable number of cDR5 were prepared and denoted as P_H-cDR5 (high valence), P_M-cDR5 (medium valence) and P_L-cDR5 (low valence). Our studies in TRAIL-sensitive and resistant cancer cell lines demonstrated that the HPMA copolymer-peptide conjugates (P-cDR5) significantly improved DR5 receptor clustering and induced apoptosis effectively. In TRAIL-sensitive colon cancer cells (COLO205, HCT-116), P-cDR5 showed efficacy comparable to anti-DR5 mAb Drozitumab (DRO), but P-cDR5 outperformed DRO in TRAIL-resistant cells (HT-29), highlighting the importance of efficient receptor clustering. In COLO205 cells P_M-cDR5 exhibited an IC50 of 94 pM, while P_H-cDR5 had an even lower IC50 of 15 pM (based on cDR5 equivalent concentration), indicating enhanced potency of the multivalent HPMA copolymer-based system with a flexible polymer backbone in comparison with the IC₅₀ for TRAIL at 0.12 nM. Combining P-cDR5 with valproic acid, a histone deacetylase inhibitor, resulted in further enhancement of apoptosis inducing efficacy, along with destabilizing mitochondrial membranes and increased sensitivity of TRAIL-resistant cells. These findings suggest that attaching multiple cDR5 peptides to a flexible water-soluble polymer carrier not only overcomes the limitations of previous designs but also offers a promising avenue for treating resistant cancers, pointing toward the need for further preclinical exploration and validation of this innovative strategy.