(W1030-04-20) Combination of Multivalent DR5 Receptor Clustering Agonists and Histone Deacetylase Inhibitors for Treatment of Colon Cancer

Purpose: Death Receptor 5 (DR5), part of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) family, is highly expressed on various tumor cells but minimally on normal cells, making it an excellent target for cancer therapies. DR5-mediated apoptosis occurs through receptor trimerization upon TRAIL binding, initiating the death-inducing signaling complex (DISC) and subsequent caspase activation (1,2). Numerous recombinant TRAIL variants and anti-death receptor antibodies were developed. However, despite activity on animal models, clinical studies failed (3). Therefore, new strategies are essential to ensure robust and safe DR5 receptor aggregation. Our aim is to develop a TRAIL-mimicking polymer-peptide conjugate (P-DR5) that promotes high-level DR5 receptor aggregation. Additionally, combining DR5-targeted therapies with valproic acid (VA), a broad-spectrum histone deacetylase inhibitor (HDACi) that destabilizes the outer mitochondrial membrane, may enhance apoptosis and sensitize TRAIL-resistant cell lines (4). To this end, the combination treatment of P-DR5 and VA on sensitive and resistant colon cancer cells was evaluated.

Methods: A polymerizable derivative of the cyclic DR5-binding peptide, MA-GGWDCLDNRIGRRQCVKL (MA is methacryloyl), was synthesized by solid-phase peptide synthesis (5). Oxidation between two cysteines was achieved by dissolving the peptide in a DMSO: ACN: 10 mM NH₄OAc solution (1:4:5 v/v) at a concentration of 1 mg/ml and stirring for 48 h. The process was monitored and the product purified by analytical reversed-phase high-performance liquid chromatography. Next, the polymerizable peptide was RAFT copolymerized with N-(2-hydroxypropyl)methacrylamide (HPMA) to yield multivalent conjugates with varying content of peptide per polymer chain: P<sub>L-DR5, P<sub>M-DR5, and P<sub>H-DR5 with valency 3, 6, and 12, respectively (Figure 1). Cy5-labeled peptide and P-DR5 conjugate were prepared by CLICK chemistry. Apoptosis induction was detected by Annexin V/PI staining followed by flow cytometry. Binding constants were determined by fluorescence assay. Cytotoxicity was measured by the CCK-8 assay; caspase 8 activity was assessed by the Promega Glo system. VA (1 mM) for 24 h was used to sensitize COLO205 cells, and 10 mM VA was used to sensitize the TRAIL-resistant HT-29 cell line.

Results: The study demonstrated the efficacy of a TRAIL-mimicking polymer-peptide conjugates (P-DR5) designed to induce apoptosis in DR5-positive human cell lines. Using COLO205, a human DR5-positive, TRAIL-sensitive cell line, the binding affinity of P<sub>M-DR5 was significantly improved compared to a free DR5-targeting peptide, with a dissociation constant (Kd) reduction from 1625 nM to 196.7 nM (Figure 2A). Higher peptide valency increased receptor aggregation efficiency, with lower IC50 values: 94 pM for P_M-DR5 and 15 pM for P_H-DR5 (Figure 2B). The free peptide and P_L-DR5 did not pose cytotoxicity at all; high apoptotic levels were already achieved with P<sub>M-DR5 indicating the importance of receptor aggregation (Figure 2C). Apoptosis mechanisms induced by P-DR5 were evaluated, with elevated caspase 8 levels indicating activation of the extrinsic apoptosis pathway (Figure 2D). Mitochondrial membrane depolarization was confirmed through JC-1 staining, and enhanced caspase 3 levels underscored the effectiveness of P-DR5 in triggering apoptosis. Drozitumab (DRO), an anti-DR5 monoclonal antibody, served as a positive control. Pretreatment of COLO205 cells with 1 mM VA for 24 h before P-DR5 treatment further augmented cell death, as evidenced by the inward-shifted curve; however, VA alone did not induce significant cell death after 24 h (Figures 2E-2F). Additionally, an increase in cytochrome C release indicated that VA activated the intrinsic apoptosis pathway (Figure 2G). In the TRAIL-resistant HT-29 cell line, P-DR5 alone induced significantly higher cell death compared to DRO, with higher doses of VA pretreatment enhancing this effect in a dose-dependent manner (Figure 3A). Additionally, high-dose VA pretreatment not only increased DR5 expression but also suggested autophagy involvement, as indicated by lysosomal membrane permeabilization and an increased LC3 protein signal, suggesting that VA activates a complex cell signaling pathway to enhance P-DR5 efficacy (Figure 3B-D).

Conclusion: This study highlights the efficacy of a novel TRAIL-mimicking polymer-peptide conjugate, P-DR5, in targeting and inducing apoptosis in DR5-expressing tumor cells. Notably, it demonstrates superior apoptosis induction compared to anti-DR5 monoclonal antibodies in TRAIL-resistant cell lines. The addition of VA further enhances this therapeutic approach, particularly in overcoming resistance in these challenging cell lines. These findings not only reaffirm the potential of P-DR5 as a powerful tool in cancer treatment but also emphasize the critical role of receptor clustering and the synergistic application of histone deacetylase inhibitors (HDACi) in optimizing therapeutic outcomes. Moving forward, these insights could pave the way for innovative cancer treatment strategies that fully leverage DR5-targeted therapies, transcending the limitations of current DR5-targeted products.

References: [1] V. Subbiah et al., Mol. Cancer Ther. 11 (2012) 2541-2546.
[2] R. Riccioni et al., Haematologica 90 (2005) 612-624.
[3] A. Dubuisson, O. Micheau, Antibodies 6 (2017).
[4] S. Fulda, Exp. Cell Res. 318 (2012) 1208-1212.
[5] Y.M. Angell et al., Peptides for Youth, Springer, 2009, pp. 101-103.

Acknowledgements: Support by NIH grant R01 CA246716 (to JK) and University of Utah Seed grant (to JY).


Figure 1 Multivalent DR5 receptor clustering agonist – HPMA copolymer grafted with multiple DR5-binding peptides. (A) Synthesis of DR5 peptide agonist and synthesis of P-DR5; (C) Schematic of clustering of DR5 receptors induced by binding of P-DR5 to DR5+ cells


Figure 2 Apoptosis Induction by P-DR5 in COLO205 Cells. (A) Enhanced peptide binding affinity of P-DR5 compared to a free DR5-targeting peptide. (B) Cytotoxicity of P-DR5 with varying peptide valences treated for 24 h. (C) Apoptosis induction by conjugates with different valences. (D) Caspase 8 activation measured using the Promega Caspase-Glo assay kit. (E) Cytotoxicity of VA treatment: Cells incubated with varying doses of VA for 24, 48, and 72 h, followed by CCK-8 reagent addition. (F) Enhanced cytotoxicity with VA pretreatment: Comparison of cells treated only with various doses of P-DR5 and those pretreated with 1 mM VA for 24 h. (G) Cytochrome C release measured post-treatment using the Thermo Fisher human cytochrome c release kit.


Figure 3 VA sensitizes the TRAIL-resistant DR5-positive colorectal cancer cell line HT-29. (A) Cell death induction: Assessed after various doses of VA pretreatment for 24 h, followed by treatment with 500 nM P-DR5 or 250 nM of DRO. (B) DR5 expression post-VA treatment (C) LC3 protein measurement using confocal microscopy. (D) Autophagy measurement with Lysotracker Green DND-26.