RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells

Zhengkui Zhang; Xiangjun Kong; Maarten A. Ligtenberg; Susan E. van Hal-van Veen; Nils L. Visser; Beaunelle de Bruijn; Kelly Stecker; Pim W. van der Helm; Thomas Kuilman; Esmée P. Hoefsmit; David W. Vredevoogd; Georgi Apriamashvili; Beau Baars; Emile E. Voest; Sjoerd Klarenbeek; Maarten Altelaar; Daniel S. Peeper

doi:10.1016/j.xcrm.2022.100655

RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells

Zhengkui Zhang, Xiangjun Kong, Maarten A. Ligtenberg, Susan E. van Hal-van Veen, Nils L. Visser, Beaunelle de Bruijn, Kelly Stecker, Pim W. van der Helm, Thomas Kuilman, Esmée P. Hoefsmit, David W. Vredevoogd, Georgi Apriamashvili, Beau Baars, Emile E. Voest, Sjoerd Klarenbeek, Maarten Altelaar, Daniel S. Peeper^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

Tumor escape mechanisms for immunotherapy include deficiencies in antigen presentation, diminishing adaptive CD8⁺ T cell antitumor activity. Although innate natural killer (NK) cells are triggered by loss of MHC class I, their response is often inadequate. To increase tumor susceptibility to both innate and adaptive immune elimination, we performed parallel genome-wide CRISPR-Cas9 knockout screens under NK and CD8⁺ T cell pressure. We identify all components, RNF31, RBCK1, and SHARPIN, of the linear ubiquitination chain assembly complex (LUBAC). Genetic and pharmacologic ablation of RNF31, an E3 ubiquitin ligase, strongly sensitizes cancer cells to NK and CD8⁺ T cell killing. This occurs in a tumor necrosis factor (TNF)-dependent manner, causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I. A small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF and greatly enhances bystander killing of MHC antigen-deficient tumor cells. These results merit exploration of RNF31 inhibition as a clinical pharmacological opportunity for immunotherapy-refractory cancers.

Original language	English
Article number	100655
Pages (from-to)	1-25
Journal	Cell Reports Medicine
Volume	3
Issue number	6
DOIs	https://doi.org/10.1016/j.xcrm.2022.100655
Publication status	Published - 21 Jun 2022

Keywords

bystander killing
function-based genome-wide screens
immunotherapy resistance
NK cells
T cells
Tumor Escape
Ubiquitination
Killer Cells, Natural
Tumor Necrosis Factor-alpha/metabolism
Ubiquitin-Protein Ligases/genetics

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Zhang, Z., Kong, X., Ligtenberg, M. A., van Hal-van Veen, S. E., Visser, N. L., de Bruijn, B., Stecker, K., van der Helm, P. W., Kuilman, T., Hoefsmit, E. P., Vredevoogd, D. W., Apriamashvili, G., Baars, B., Voest, E. E., Klarenbeek, S., Altelaar, M., & Peeper, D. S. (2022). RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells. Cell Reports Medicine, 3(6), 1-25. Article 100655. https://doi.org/10.1016/j.xcrm.2022.100655

@article{4b75381e53e54e72871d46e896e26ffb,

title = "RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells",

abstract = "Tumor escape mechanisms for immunotherapy include deficiencies in antigen presentation, diminishing adaptive CD8+ T cell antitumor activity. Although innate natural killer (NK) cells are triggered by loss of MHC class I, their response is often inadequate. To increase tumor susceptibility to both innate and adaptive immune elimination, we performed parallel genome-wide CRISPR-Cas9 knockout screens under NK and CD8+ T cell pressure. We identify all components, RNF31, RBCK1, and SHARPIN, of the linear ubiquitination chain assembly complex (LUBAC). Genetic and pharmacologic ablation of RNF31, an E3 ubiquitin ligase, strongly sensitizes cancer cells to NK and CD8+ T cell killing. This occurs in a tumor necrosis factor (TNF)-dependent manner, causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I. A small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF and greatly enhances bystander killing of MHC antigen-deficient tumor cells. These results merit exploration of RNF31 inhibition as a clinical pharmacological opportunity for immunotherapy-refractory cancers.",

keywords = "bystander killing, function-based genome-wide screens, immunotherapy resistance, NK cells, T cells, Tumor Escape, Ubiquitination, Killer Cells, Natural, Tumor Necrosis Factor-alpha/metabolism, Ubiquitin-Protein Ligases/genetics",

author = "Zhengkui Zhang and Xiangjun Kong and Ligtenberg, {Maarten A.} and {van Hal-van Veen}, {Susan E.} and Visser, {Nils L.} and {de Bruijn}, Beaunelle and Kelly Stecker and {van der Helm}, {Pim W.} and Thomas Kuilman and Hoefsmit, {Esm{\'e}e P.} and Vredevoogd, {David W.} and Georgi Apriamashvili and Beau Baars and Voest, {Emile E.} and Sjoerd Klarenbeek and Maarten Altelaar and Peeper, {Daniel S.}",

note = "Funding Information: We thank all members of the Peeper laboratory for helpful discussions. We thank the Animal facility, Cytometry facility, and Genomics core facility of NKI for support. We thank Joleen Traets and Alex van Vliet for help with bioinformatic analyses, and Disha Rao for help with FACS analysis. This work was supported by LSH-TKI grant LSHM20019, Oncode TDF grant P2019-0060, and Oncode base funding to D.S.P. X.K. M.A.L. and D.S.P. conceived the project. X.K. and M.A.L. performed the screen. Z.Z. and T.K. performed bioinformatic analyses. M.A.L. B.d.B. and S.E.v.H.-v.V. performed in vivo experiments. P.W.v.d.H. E.E.V. and Z.Z. were responsible for organoids experiments. Z.Z. and X.K. performed all other experiments. K.E.S. and M.A. performed mass spectrometric analysis. S.K. performed the IHC quantification. N.L.V. S.E.v.H.-v.V. E.P.H. D.W.V. G.A. and B.B. provided technical support. G.A. assisted with statistical analyses. Z.Z. X.K. and D.S.P. wrote the manuscript. All authors revised and approved the manuscript. The project was supervised by D.S.P. D.S.P. is co-founder, shareholder, and advisor of Immagene. M.A.L. is co-founder, shareholder, and CEO of Immagene. The other authors declare no competing interests. Funding Information: We thank all members of the Peeper laboratory for helpful discussions. We thank the Animal facility, Cytometry facility, and Genomics core facility of NKI for support. We thank Joleen Traets and Alex van Vliet for help with bioinformatic analyses, and Disha Rao for help with FACS analysis. This work was supported by LSH -TKI grant LSHM20019 , Oncode TDF grant P2019-0060 , and Oncode base funding to D.S.P. Publisher Copyright: {\textcopyright} 2022 Netherlands Cancer Institute Copyright {\textcopyright} 2022 Netherlands Cancer Institute. Published by Elsevier Inc. All rights reserved.",

year = "2022",

month = jun,

day = "21",

doi = "10.1016/j.xcrm.2022.100655",

language = "English",

volume = "3",

pages = "1--25",

number = "6",

}

Zhang, Z, Kong, X, Ligtenberg, MA, van Hal-van Veen, SE, Visser, NL, de Bruijn, B, Stecker, K, van der Helm, PW, Kuilman, T, Hoefsmit, EP, Vredevoogd, DW, Apriamashvili, G, Baars, B, Voest, EE, Klarenbeek, S, Altelaar, M & Peeper, DS 2022, 'RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells', Cell Reports Medicine, vol. 3, no. 6, 100655, pp. 1-25. https://doi.org/10.1016/j.xcrm.2022.100655

TY - JOUR

T1 - RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells

AU - Zhang, Zhengkui

AU - Kong, Xiangjun

AU - Ligtenberg, Maarten A.

AU - van Hal-van Veen, Susan E.

AU - Visser, Nils L.

AU - de Bruijn, Beaunelle

AU - Stecker, Kelly

AU - van der Helm, Pim W.

AU - Kuilman, Thomas

AU - Hoefsmit, Esmée P.

AU - Vredevoogd, David W.

AU - Apriamashvili, Georgi

AU - Baars, Beau

AU - Voest, Emile E.

AU - Klarenbeek, Sjoerd

AU - Altelaar, Maarten

AU - Peeper, Daniel S.

N1 - Funding Information: We thank all members of the Peeper laboratory for helpful discussions. We thank the Animal facility, Cytometry facility, and Genomics core facility of NKI for support. We thank Joleen Traets and Alex van Vliet for help with bioinformatic analyses, and Disha Rao for help with FACS analysis. This work was supported by LSH-TKI grant LSHM20019, Oncode TDF grant P2019-0060, and Oncode base funding to D.S.P. X.K. M.A.L. and D.S.P. conceived the project. X.K. and M.A.L. performed the screen. Z.Z. and T.K. performed bioinformatic analyses. M.A.L. B.d.B. and S.E.v.H.-v.V. performed in vivo experiments. P.W.v.d.H. E.E.V. and Z.Z. were responsible for organoids experiments. Z.Z. and X.K. performed all other experiments. K.E.S. and M.A. performed mass spectrometric analysis. S.K. performed the IHC quantification. N.L.V. S.E.v.H.-v.V. E.P.H. D.W.V. G.A. and B.B. provided technical support. G.A. assisted with statistical analyses. Z.Z. X.K. and D.S.P. wrote the manuscript. All authors revised and approved the manuscript. The project was supervised by D.S.P. D.S.P. is co-founder, shareholder, and advisor of Immagene. M.A.L. is co-founder, shareholder, and CEO of Immagene. The other authors declare no competing interests. Funding Information: We thank all members of the Peeper laboratory for helpful discussions. We thank the Animal facility, Cytometry facility, and Genomics core facility of NKI for support. We thank Joleen Traets and Alex van Vliet for help with bioinformatic analyses, and Disha Rao for help with FACS analysis. This work was supported by LSH -TKI grant LSHM20019 , Oncode TDF grant P2019-0060 , and Oncode base funding to D.S.P. Publisher Copyright: © 2022 Netherlands Cancer Institute Copyright © 2022 Netherlands Cancer Institute. Published by Elsevier Inc. All rights reserved.

PY - 2022/6/21

Y1 - 2022/6/21

N2 - Tumor escape mechanisms for immunotherapy include deficiencies in antigen presentation, diminishing adaptive CD8+ T cell antitumor activity. Although innate natural killer (NK) cells are triggered by loss of MHC class I, their response is often inadequate. To increase tumor susceptibility to both innate and adaptive immune elimination, we performed parallel genome-wide CRISPR-Cas9 knockout screens under NK and CD8+ T cell pressure. We identify all components, RNF31, RBCK1, and SHARPIN, of the linear ubiquitination chain assembly complex (LUBAC). Genetic and pharmacologic ablation of RNF31, an E3 ubiquitin ligase, strongly sensitizes cancer cells to NK and CD8+ T cell killing. This occurs in a tumor necrosis factor (TNF)-dependent manner, causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I. A small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF and greatly enhances bystander killing of MHC antigen-deficient tumor cells. These results merit exploration of RNF31 inhibition as a clinical pharmacological opportunity for immunotherapy-refractory cancers.

AB - Tumor escape mechanisms for immunotherapy include deficiencies in antigen presentation, diminishing adaptive CD8+ T cell antitumor activity. Although innate natural killer (NK) cells are triggered by loss of MHC class I, their response is often inadequate. To increase tumor susceptibility to both innate and adaptive immune elimination, we performed parallel genome-wide CRISPR-Cas9 knockout screens under NK and CD8+ T cell pressure. We identify all components, RNF31, RBCK1, and SHARPIN, of the linear ubiquitination chain assembly complex (LUBAC). Genetic and pharmacologic ablation of RNF31, an E3 ubiquitin ligase, strongly sensitizes cancer cells to NK and CD8+ T cell killing. This occurs in a tumor necrosis factor (TNF)-dependent manner, causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I. A small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF and greatly enhances bystander killing of MHC antigen-deficient tumor cells. These results merit exploration of RNF31 inhibition as a clinical pharmacological opportunity for immunotherapy-refractory cancers.

KW - bystander killing

KW - function-based genome-wide screens

KW - immunotherapy resistance

KW - NK cells

KW - T cells

KW - Tumor Escape

KW - Ubiquitination

KW - Killer Cells, Natural

KW - Tumor Necrosis Factor-alpha/metabolism

KW - Ubiquitin-Protein Ligases/genetics

UR - http://www.scopus.com/inward/record.url?scp=85133101269&partnerID=8YFLogxK

U2 - 10.1016/j.xcrm.2022.100655

DO - 10.1016/j.xcrm.2022.100655

M3 - Article

C2 - 35688159

AN - SCOPUS:85133101269

SN - 2666-3791

VL - 3

SP - 1

EP - 25

JO - Cell Reports Medicine

JF - Cell Reports Medicine

IS - 6

M1 - 100655

ER -

RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells

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