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HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy

HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy
HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy
Background
Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb.

Methods
We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription.

Results
We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes.

Conclusion
Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.
Animals, Antibodies, Monoclonal/pharmacology, Humans, Hypoxia/metabolism, Immunotherapy, Leukemia, Lymphocytic, Chronic, B-Cell/metabolism, Macrophages/metabolism, Mice, Receptors, IgG/genetics, Tumor Microenvironment
0392-9078
131
Hussain, Khiyam
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Liu, Rena
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Smith, Rosanna C G
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Müller, Kri T J
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Ghorbani, Mohammadmersad
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Macari, Sofia
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Cleary, Kirstie L S
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Oldham, Robert J
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Foxall, Russell B
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James, Sonya
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Booth, Steven G
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Murray, Tom
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Dahal, Lekh N
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Hargreaves, Chantal E
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Kemp, Robert S
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Longley, Jemma
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Douglas, James
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Markham, Hannah
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Chee, Serena J
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Stopforth, Richard J
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Roghanian, Ali
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Carter, Matthew J
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Ottensmeier, Christian H
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Frendéus, Bjorn
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Cutress, Ramsey I
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French, Ruth R
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Glennie, Martin J
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Strefford, Jonathan C
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Thirdborough, Stephen M
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Beers, Stephen A
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Cragg, Mark S
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Hussain, Khiyam
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Liu, Rena
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Smith, Rosanna C G
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Müller, Kri T J
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Ghorbani, Mohammadmersad
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Macari, Sofia
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Cleary, Kirstie L S
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Oldham, Robert J
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Foxall, Russell B
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James, Sonya
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Booth, Steven G
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Murray, Tom
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Dahal, Lekh N
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Hargreaves, Chantal E
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Kemp, Robert S
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Longley, Jemma
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Douglas, James
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Markham, Hannah
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Chee, Serena J
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Stopforth, Richard J
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Roghanian, Ali
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Frendéus, Bjorn
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Cutress, Ramsey I
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French, Ruth R
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Glennie, Martin J
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Strefford, Jonathan C
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Thirdborough, Stephen M
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Beers, Stephen A
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Cragg, Mark S
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Hussain, Khiyam, Liu, Rena, Smith, Rosanna C G, Müller, Kri T J, Ghorbani, Mohammadmersad, Macari, Sofia, Cleary, Kirstie L S, Oldham, Robert J, Foxall, Russell B, James, Sonya, Booth, Steven G, Murray, Tom, Dahal, Lekh N, Hargreaves, Chantal E, Kemp, Robert S, Longley, Jemma, Douglas, James, Markham, Hannah, Chee, Serena J, Stopforth, Richard J, Roghanian, Ali, Carter, Matthew J, Ottensmeier, Christian H, Frendéus, Bjorn, Cutress, Ramsey I, French, Ruth R, Glennie, Martin J, Strefford, Jonathan C, Thirdborough, Stephen M, Beers, Stephen A and Cragg, Mark S (2022) HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy. Journal of Experimental and Clinical Cancer Research, 41 (131), 131, [131]. (doi:10.1186/s13046-022-02294-5).

Record type: Article

Abstract

Background
Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb.

Methods
We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription.

Results
We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes.

Conclusion
Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

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Accepted/In Press date: 20 February 2022
Published date: 7 April 2022
Additional Information: Funding Information: Funding was provided by Cancer Research UK, programme grant awarded to M.S.C., J.C.S., S.M.T and S.A.B. (Award number: A24721) and the United Kingdom National Centre for the Replacement, Refinement and Reduction of Animals in Research, CRACKIT Programme grant awarded to M.J.G., S.A.B. and A.R. (Award number: NC3Rs 15402–106217) and Cancer Research UK and Experimental Cancer Centre awards C328/A25139 and C24563/A25171. TM, RL, KTJM and JL were funded through Cancer Research UK studentship award C328/A25169. TM was also funded through the Cancer Immunology Talent fund. RJS was funded by BBSRC iCASE award with Promega. Funding Information: We would like to thank the members of the Antibody and Vaccine group for useful discussions and the pre-clinical unit staff for animal husbandry and assistance. ATAC-seq samples were prepared by Victoria Gernedl in the Bock lab and bioinformatically processed by Bekir Erg?ner. The sequencing was performed by the Biomedical Sequencing Facility at the Center for Molecular Medicine, Vienna. We would like to thank Dr. Francesco Forconi, Dr. N Kathleen Potter, Dr. Ian Tracy and Mrs. Isla Henderson (University of Southampton, The Human Tissue Bank) for provision of CLL samples. We would like to thank Ms. Deborah Donovan (Southampton NHS Trust) for assisting us in the use of the ABL 835 FLEX analyzer, in order to measure metabolites and electrolytes in cell culture supernatants. We would like to thank Prof J and D Mann (University of Newcastle) for insightful initial discussions. We would like to thank Dr Zo? S Walters and Dr Ian Tracy for their expertise on ChIP assays. We would like to thank CRUK Southampton Centre Bioinformatics core facility for supporting this work. Finally, we would like to acknowledge Dr Charles Tilley and Dr Janlyn Falconer for assistance with pathology and provision of primary tumor clinical samples, respectively. Funding Information: A.R. receives funding from BioInvent International. Research by R.I.C is supported by use of equipment to measure body composition provided by SECA through a model industry collaborative agreement (mICA) with University Hospital Southampton. M.J.G previously acted as a consultant to a number of biotech companies and receives institutional payments and royalties from antibody patents and licenses. J.C.S has received funding from Roche. S.A.B acts as a consultant for a number of biotech companies and has received institutional support for grants and patents from BioInvent. M.S.C. acts as a consultant for a number of biotech companies, being retained as a consultant for BioInvent International and has received research funding from BioInvent, GSK, UCB, iTeos, and Roche. Publisher Copyright: © 2022, The Author(s).
Keywords: Animals, Antibodies, Monoclonal/pharmacology, Humans, Hypoxia/metabolism, Immunotherapy, Leukemia, Lymphocytic, Chronic, B-Cell/metabolism, Macrophages/metabolism, Mice, Receptors, IgG/genetics, Tumor Microenvironment

Identifiers

Local EPrints ID: 456881
URI: http://eprints.soton.ac.uk/id/eprint/456881
ISSN: 0392-9078
PURE UUID: 3df779fe-a7e4-4ac7-b4e2-fa259a7f40b1
ORCID for Kri T J Müller: ORCID iD orcid.org/0000-0001-5674-6125
ORCID for Sofia Macari: ORCID iD orcid.org/0000-0002-6506-4051
ORCID for Robert J Oldham: ORCID iD orcid.org/0000-0002-8007-1145
ORCID for Ali Roghanian: ORCID iD orcid.org/0000-0003-1316-4218
ORCID for Jonathan C Strefford: ORCID iD orcid.org/0000-0002-0972-2881
ORCID for Stephen A Beers: ORCID iD orcid.org/0000-0002-3765-3342
ORCID for Mark S Cragg: ORCID iD orcid.org/0000-0003-2077-089X

Catalogue record

Date deposited: 16 May 2022 16:30
Last modified: 11 Jul 2024 01:57

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Contributors

Author: Khiyam Hussain
Author: Rena Liu
Author: Rosanna C G Smith
Author: Kri T J Müller ORCID iD
Author: Mohammadmersad Ghorbani
Author: Sofia Macari ORCID iD
Author: Kirstie L S Cleary
Author: Robert J Oldham ORCID iD
Author: Russell B Foxall
Author: Sonya James
Author: Steven G Booth
Author: Tom Murray
Author: Lekh N Dahal
Author: Chantal E Hargreaves
Author: Robert S Kemp
Author: Jemma Longley
Author: James Douglas
Author: Hannah Markham
Author: Serena J Chee
Author: Richard J Stopforth
Author: Ali Roghanian ORCID iD
Author: Matthew J Carter
Author: Bjorn Frendéus
Author: Ruth R French
Author: Stephen A Beers ORCID iD
Author: Mark S Cragg ORCID iD

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