A patch of positively charged residues regulates the efficacy of clinical DR5 antibodies in solid tumors

Shivange, Gururaj; Mondal, Tanmoy; Lyerly, Evan; Bhatnagar, Sanchita; Landen, Charles N.; Reddy, Shivani; Kim, Jonathan; Doan, Britney; Riddle, Paula; Tushir-Singh, Jogender

doi:10.1016/j.celrep.2021.109953

Cited by 5 publications

(33 citation statements)

References 41 publications

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“…However, the relationship between the DDA-induced ER stress response and DDA effects on DR5 were unexplored. The results presented here show that individual mutational disruption of a total of five of the seven DR5 disulfide bonds each causes the same DR5 stabilization, including the disulfides within the previously described auto-inhibitory domain [24, 25]. The observation that DDAs still induce a mobility shift of monomeric disulfide point mutants of DR5 suggests that while loss of individual disulfide bonds is sufficient to stabilize DR5 and promote pro-apoptotic signaling, DDAs disrupt multiple DR5 disulfide bonds.…”

Section: Discussionsupporting

confidence: 53%

“…A recent study showed that the disulfide bond-rich extracellular domain of DR5 serves to prevent receptor oligomerization and pro-apoptotic signaling in the absence of its ligand TRAIL [24]. A more recent article narrowed down the DR5 autoinhibitory domain to a positive patch involving residues R154, K155, and R157 [25]. Based on a crystal structure [26], these basic residues share a common orientation due to two disulfide bonds, C156-C170 and C139-153 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted March 7, 2024. ; https://doi.org/10.1101/2024.03.04.583390 doi: bioRxiv preprint [25]. Based on a crystal structure [26], these basic residues share a common orientation due to two disulfide bonds, C156-C170 and C139-153 (Fig.…”

Section: Dr5 Levels and Oligomerization States Are Altered By Perturb...mentioning

confidence: 99%

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DR5 disulfide bonding as a sensor and effector of protein folding stress

Law,

Dulloo,

Eggleston

et al. 2024

Preprint

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New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.

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Section: Discussionsupporting

confidence: 53%

Section: Resultsmentioning

confidence: 99%

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DR5 disulfide bonding as a sensor and effector of protein folding stress

Law,

Dulloo,

Eggleston

et al. 2024

Preprint

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“…1F and G ). Next, based on a range of PiTou scores, various Fc-extended peptides were inserted into recombinant death receptor-5, rDR5-IgG4-Fc ( 20 ) by replacing the region near its patch of positively charged residue motif (RKCR) ( 21 ) in the third cysteine-rich domain ( Fig. 1H and I , see the sequences in blue, red, and black).…”

Section: Resultsmentioning

confidence: 99%

A Feasible Alternative Strategy Targeting Furin Disrupts SARS-CoV-2 Infection Cycle

et al. 2022

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“…However, the clustering mechanisms for both DR5 and Fas remained elusive until recently. Using clinical bivalent or multivalent monospecific antibodies, bispecific antibodies, receptor mutagenesis studies, functional assays, and NMR structure analysis of DR5, a series of papers have discovered and characterized receptor clustering conformation autoinhibitory motifs in the DR5 extracellular domain (ECD) [3,4]. The key described motif comprises a cysteine-stabilized patch of positively charged (arginine, lysine) amino acid residues in DR5 CRD3.…”

Section: Introductionmentioning

confidence: 99%

Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer

Mondal,

Gaur,

Wamba

et al. 2023

Cell Death Differ

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Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors.

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A patch of positively charged residues regulates the efficacy of clinical DR5 antibodies in solid tumors

Cited by 5 publications

References 41 publications

DR5 disulfide bonding as a sensor and effector of protein folding stress

DR5 disulfide bonding as a sensor and effector of protein folding stress

A Feasible Alternative Strategy Targeting Furin Disrupts SARS-CoV-2 Infection Cycle

Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer

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