Correction: Yagolovich et al. DR5-Selective TRAIL Variant DR5-B Functionalized with Tumor-Penetrating iRGD Peptide for Enhanced Antitumor Activity against Glioblastoma. Int. J. Mol. Sci. 2022, 23, 12687.

In the original publication [...].

Dual targeting of DR5 and VEGFR2 molecular pathways by multivalent fusion protein significantly suppresses tumor growth and angiogenesis.

Destroying tumor vasculature is a relevant therapeutic strategy due to its involvement in tumor progression. However, adaptive resistance to approved antiangiogenic drugs targeting VEGF/VEGFR pathway requires the recruitment of additional targets. In this aspect, targeting TRAIL pathway is promising as it is an important component of the immune system involved in tumor immunosurveillance. For dual targeting of malignant cells and tumor vascular microenvironment, we designed a multivalent fusion protein SRH-DR5-B-iRGD with antiangiogenic VEGFR2-specific peptide SRH at the N-terminus and a tumor-targeting and -penetrating peptide iRGD at the C-terminus of receptor-selective TRAIL variant DR5-B. SRH-DR5-B-iRGD obtained high affinity for DR5, VEGFR2 and αvß3 integrin in nanomolar range. Fusion of DR5-B with effector peptides accelerated DR5 receptor internalization rate upon ligand binding. Antitumor efficacy was evaluated in vitro in human tumor cell lines and primary patient-derived glioblastoma neurospheres, and in vivo in xenograft mouse model of human glioblastoma. Multivalent binding of SRH-DR5-B-iRGD fusion efficiently stimulated DR5-mediated tumor cell death via caspase-dependent mechanism, suppressed xenograft tumor growth by >80 %, doubled the lifespan of xenograft animals, and inhibited tumor vascularization. Therefore, targeting DR5 and VEGFR2 molecular pathways with SRH-DR5-B-iRGD protein may provide a novel therapeutic approach for treatment of solid tumors.

Application of an Autoinduction Strategy to Optimize the Heterologous Production of an Antitumor Bispecific Fusion Protein Based on the TRAIL Receptor-Selective Mutant Variant in Escherichia coli.

Autoinduction is a simple approach for heterologous protein expression that helps to achieve the high-level production of recombinant proteins in soluble form. In this work, we investigated if the application of an autoinduction strategy could help to optimize the production of bifunctional protein SRH-DR5-B, the DR5-specific TRAIL variant DR5-B fused to a VEGFR2-specific peptide SRHTKQRHTALH for dual antitumor and antiangiogenic activity. The protein was expressed in Escherichia coli SHuffle B T7, BL21(DE3), and BL21(DE3)pLysS strains. By IPTG induction, the highest expression level was in SHuffle B T7, while by autoinduction, the similar expression level was achieved in BL21(DE3)pLysS. However, in SHuffle B T7, only 45% of IPTG-induced SRH-DR5-B was expressed in soluble form, in contrast to 75% autoinduced in BL21(DE3)pLysS. The yield of purified SRH-DR5-B protein expressed by autoinduction in BL21(DE3)pLysS was 28 ± 4.5 mg per 200 ml of cell culture, which was 1.4 times higher than the yield from IPTG-induced SHuffle B T7. Regardless of the production method, SRH-DR5-B was equally cytotoxic to BxPC-3 human tumor cells expressing DR5 and VEGFR2 receptors. Thus, the production of SRH-DR5-B by autoinduction in the E. coli BL21(DE3)pLysS strain is an efficient, technologically simple, and economical technique that allows to obtain a large amount of active protein from the cytoplasmic cell fraction. Our work demonstrates that the strategy of induction of protein expression is no less important than the strain selection.

ONC201-Induced Mitochondrial Dysfunction, Senescence-like Phenotype, and Sensitization of Cultured BT474 Human Breast Cancer Cells to TRAIL.

ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

DR5-Selective TRAIL Variant DR5-B Functionalized with Tumor-Penetrating iRGD Peptide for Enhanced Antitumor Activity against Glioblastoma.

TRAIL (TNF-related apoptosis-inducing ligand) and its derivatives are potentials for anticancer therapy due to the selective induction of apoptosis in tumor cells upon binding to death receptors DR4 or DR5. Previously, we generated a DR5-selective TRAIL mutant variant DR5-B overcoming receptor-dependent resistance of tumor cells to TRAIL. In the current study, we improved the antitumor activity of DR5-B by fusion with a tumor-homing iRGD peptide, which is known to enhance the drug penetration into tumor tissues. The obtained bispecific fusion protein DR5-B-iRGD exhibited dual affinity for DR5 and integrin αvß3 receptors. DR5-B-iRGD penetrated into U-87 tumor spheroids faster than DR5-B and demonstrated an enhanced antitumor effect in human glioblastoma cell lines T98G and U-87, as well as in primary patient-derived glioblastoma neurospheres in vitro. Additionally, DR5-B-iRGD was highly effective in a xenograft mouse model of the U-87 human glioblastoma cell line in vivo. We suggest that DR5-B-iRGD may become a promising candidate for targeted therapy for glioblastoma.

Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH-DR5-B with Dual VEGFR2 and DR5 Receptor Specificity.

In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH-DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH-DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH-DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH-DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH-DR5-B can be considered as a promising candidate for therapeutic applications.

Death Receptors DR4 and DR5 Undergo Spontaneous and Ligand-Mediated Endocytosis and Recycling Regardless of the Sensitivity of Cancer Cells to TRAIL.

Tumor necrosis factor-associated ligand inducing apoptosis (TRAIL) induces apoptosis through the death receptors (DRs) 4 and 5 expressed on the cell surface. Upon ligand stimulation, death receptors are rapidly internalized through clathrin-dependent and -independent mechanisms. However, there have been conflicting data on the role of death receptor endocytosis in apoptotic TRAIL signaling and possible cell type-specific differences in TRAIL signaling have been proposed. Here we have compared the kinetics of TRAIL-mediated internalization and subsequent recycling of DR4 and DR5 in resistant (HT-29 and A549) and sensitive (HCT116 and Jurkat) tumor cell lines of various origin. TRAIL stimulated the internalization of both receptors in a concentration-dependent manner with similar kinetics in sensitive and resistant cell lines without affecting the steady-state expression of DR4 and DR5 in cell lysates. Using the receptor-selective TRAIL variant DR5-B, we have shown that DR5 is internalized independently of DR4 receptor. After internalization and elimination of TRAIL from culture medium, the receptors slowly return to the plasma membrane. Within 4 h in resistant or 6 h in sensitive cells, the surface expression of receptors was completely restored. Recovery of receptors occurred both from newly synthesized molecules or from trans-Golgi network, as cycloheximide and brefeldin A inhibited this process. These agents also suppressed the expression of cell surface receptors in a time- and concentration-dependent manner, indicating that DRs undergo constitutive endocytosis. Inhibition of receptor endocytosis by sucrose led to sensitization of resistant cells to TRAIL and to an increase in its cytotoxic activity against sensitive cells. Our results confirm the universal nature of TRAIL-induced death receptor endocytosis, thus cell sensitivity to TRAIL can be associated with post-endocytic events.

Amphiphilic Poly(N-vinylpyrrolidone) Nanoparticles Conjugated with DR5-Specific Antitumor Cytokine DR5-B for Targeted Delivery to Cancer Cells.

Nanoparticles based on the biocompatible amphiphilic poly(N-vinylpyrrolidone) (Amph-PVP) derivatives are promising for drug delivery. Amph-PVPs self-aggregate in aqueous solutions with the formation of micellar nanoscaled structures. Amph-PVP nanoparticles are able to immobilize therapeutic molecules under mild conditions. As is well known, many efforts have been made to exploit the DR5-dependent apoptosis induction for cancer treatment. The aim of the study was to fabricate Amph-PVP-based nanoparticles covalently conjugated with antitumor DR5-specific TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) variant DR5-B and to evaluate their in vitro cytotoxicity in 3D tumor spheroids. The Amph-PVP nanoparticles were obtained from a 1:1 mixture of unmodified and maleimide-modified polymeric chains, while DR5-B protein was modified by cysteine residue at the N-end for covalent conjugation with Amph-PVP. The nanoparticles were found to enhance cytotoxicity effects compared to those of free DR5-B in both 2D (monolayer culture) and 3D (tumor spheroids) in vitro models. The cytotoxicity of the nanoparticles was investigated in human cell lines, namely breast adenocarcinoma MCF-7 and colorectal carcinomas HCT116 and HT29. Notably, DR5-B conjugation with Amph-PVP nanoparticles sensitized resistant multicellular tumor spheroids from MCF-7 and HT29 cells. Taking into account the nanoparticles loading ability with a wide range of low-molecular-weight antitumor chemotherapeutics into hydrophobic core and feasibility of conjugation with hydrophilic therapeutic molecules by click chemistry, we suggest further development to obtain a versatile system for targeted drug delivery into tumor cells.

Corrigendum: Death Receptors DR4 and DR5 Undergo Spontaneous and Ligand-Mediated Endocytosis and Recycling Regardless of the Sensitivity of Cancer Cells to TRAIL.

[This corrects the article DOI: 10.3389/fcell.2021.733688.].

Genetically Modified DR5-Specific TRAIL Variant DR5-B Revealed Dual Antitumor and Protumoral Effect in Colon Cancer Xenografts and an Improved Pharmacokinetic Profile.

Despite the weak clinical efficacy of TRAIL death receptor agonists, a search is under way for new agents that more efficiently activate apoptotic signaling. We previously created a TRAIL DR5-selective variant DR5-B without affinity for the DR4, DcR1, DcR2, and OPG receptors and increased proapoptotic activity in tumor cells. Here we showed that DR5-B significantly inhibited tumor growth in HCT116 and Caco-2 but not in HT-29 xenografts. The antitumor activity of DR5-B was 2.5 times higher in HCT116 xenografts compared to TRAIL. DR5-B at a dose of 2 or 10 mg/kg/d for 10 days inhibited tumor growth in HCT116 xenografts by 26% or 50% respectively, and increased animal survival. Unexpectedly, DR5-B at a higher dose (25 mg/kg/d) inhibited tumor growth only during the first 8 days of drug exposure, while at the end of the monitoring, no effect or even slight stimulation of tumor growth was observed. The pharmacokinetic parameters of DR5-B were comparable to those of TRAIL, except that the half-life was 3.5 times higher. Thus, enhancing TRAIL selectivity to DR5 may increase both antitumor and proliferative activities depending on the concentration and administration regimens.