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Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells

Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells
Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells

Background: rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours. 

Results: we demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS. 

Conclusions: the results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

Apoptosis, Cell Differentiation, Cell Line, Tumor, Child, Enhancer of Zeste Homolog 2 Protein/metabolism, Enzyme Inhibitors/pharmacology, Histone Methylation, Humans, Rhabdomyosarcoma, Tretinoin/pharmacology, Epigenetic therapy, Differentiation therapy, All-trans retinoic acid, EZH2
1868-7075
O'Brien, Eleanor
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Tse, Carmen
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Tracy, Ian
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Reddin, Ian
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Selfe, Joanna
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Gibson, Jane
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Tapper, William
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Pengelly, Reuben J.
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Gao, Jinhui
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Aladowicz, Ewa
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Petts, Gemma
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Thway, Khin
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Popov, Sergey
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Kelsey, Anna
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Underwood, Timothy J.
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Shipley, Janet
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Walters, Zoë S.
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O'Brien, Eleanor
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Tse, Carmen
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Tracy, Ian
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Reddin, Ian
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Selfe, Joanna
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Gibson, Jane
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Tapper, William
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Pengelly, Reuben J.
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Gao, Jinhui
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Aladowicz, Ewa
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Petts, Gemma
ca986029-d160-4ee0-9207-fd7ac68d70ae
Thway, Khin
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Popov, Sergey
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Kelsey, Anna
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Underwood, Timothy J.
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Shipley, Janet
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Walters, Zoë S.
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O'Brien, Eleanor, Tse, Carmen, Tracy, Ian, Reddin, Ian, Selfe, Joanna, Gibson, Jane, Tapper, William, Pengelly, Reuben J., Gao, Jinhui, Aladowicz, Ewa, Petts, Gemma, Thway, Khin, Popov, Sergey, Kelsey, Anna, Underwood, Timothy J., Shipley, Janet and Walters, Zoë S. (2023) Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells. Clinical Epigenetics, 15 (1), [167]. (doi:10.1186/s13148-023-01583-w).

Record type: Article

Abstract

Background: rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours. 

Results: we demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS. 

Conclusions: the results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.

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Accepted/In Press date: 9 October 2023
e-pub ahead of print date: 19 October 2023
Published date: December 2023
Additional Information: Funding Information: This work was funded by Children with Cancer UK [grant 14-177], Sarcoma UK [grant SUK206.2017] and The Schottlander Research Charitable Trust. Publisher Copyright: © 2023, BioMed Central Ltd., part of Springer Nature.
Keywords: Apoptosis, Cell Differentiation, Cell Line, Tumor, Child, Enhancer of Zeste Homolog 2 Protein/metabolism, Enzyme Inhibitors/pharmacology, Histone Methylation, Humans, Rhabdomyosarcoma, Tretinoin/pharmacology, Epigenetic therapy, Differentiation therapy, All-trans retinoic acid, EZH2

Identifiers

Local EPrints ID: 483293
URI: http://eprints.soton.ac.uk/id/eprint/483293
ISSN: 1868-7075
PURE UUID: f3d92e95-394e-46c1-a205-ad35c3ce4af0
ORCID for Ian Tracy: ORCID iD orcid.org/0000-0003-4624-672X
ORCID for Ian Reddin: ORCID iD orcid.org/0000-0001-5478-7855
ORCID for William Tapper: ORCID iD orcid.org/0000-0002-5896-1889
ORCID for Reuben J. Pengelly: ORCID iD orcid.org/0000-0001-7022-645X
ORCID for Jinhui Gao: ORCID iD orcid.org/0000-0001-9588-7561
ORCID for Timothy J. Underwood: ORCID iD orcid.org/0000-0001-9455-2188
ORCID for Zoë S. Walters: ORCID iD orcid.org/0000-0002-1835-5868

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Date deposited: 27 Oct 2023 16:40
Last modified: 15 Aug 2024 02:17

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Contributors

Author: Eleanor O'Brien
Author: Carmen Tse
Author: Ian Tracy ORCID iD
Author: Ian Reddin ORCID iD
Author: Joanna Selfe
Author: Jane Gibson
Author: William Tapper ORCID iD
Author: Jinhui Gao ORCID iD
Author: Ewa Aladowicz
Author: Gemma Petts
Author: Khin Thway
Author: Sergey Popov
Author: Anna Kelsey
Author: Janet Shipley
Author: Zoë S. Walters ORCID iD

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