Epigenetic therapy suppresses endocrine-resistant breast tumour growth by re-wiring ER-mediated 3D chromatin interactions

TLDR: In this paper, the authors show that treatment of endocrine-resistant estrogen receptor positive (ER+) breast cancer with an FDA-approved epigenetic therapy Decitabine (5-Aza-mC), results in genome-wide DNA hypomethylation and suppression of tumour growth in preclinical metastatic ER+ breast tumour xenograft models.

Abstract: Three-dimensional (3D) epigenome remodelling is emerging as an important mechanism of gene deregulation in cancer. However, its potential as a target to overcome cancer therapy resistance remains largely unaddressed. Here we show that treatment of endocrine-resistant estrogen receptor positive (ER+) breast cancer with an FDA-approved epigenetic therapy Decitabine (5-Aza-mC), results in genome-wide DNA hypomethylation and suppression of tumour growth in preclinical metastatic ER+ breast tumour xenograft models. Systematic integration of matched chromatin conformation capture (Hi-C), Promoter Capture Hi-C, RNA-seq and ER ChIP-seq data revealed widespread effects on epigenome deregulation, including de-compaction of higher order chromatin structure and loss of topologically associating domains (TAD) boundary insulation. Key enhancer ER binding sites were demethylated and re-activated after Decitabine treatment, resulting in new ER-mediated enhancer-promoter interactions and concordant activation of tumour-suppressive gene pathways. Importantly, we show that the activated ER target genes were also predictive of good outcome in multiple ER+ breast cancer clinical cohorts. Together our study reveals a previously undescribed mechanism of Decitabine in re-wiring DNA methylation-dependent 3D genome architecture resulting in suppression of tumour growth, and highlights the potential of epigenetic therapy in targeting ER+ endocrine-resistant breast cancer. One line summaryEpigenetic therapy suppresses tumour growth in metastatic ER+ breast cancer via DNA hypomethylation-induced rewiring of long-range ER-mediated enhancer-promoter chromatin interactions to activate ER target genes involved in repression of proliferation and the cell cycle.

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1
Epigenetic therapy suppresses endocrine-resistant breast tumour growth by re-1
wiring ER-mediated 3D chromatin interactions 2
3
Authors 4
Joanna Achinger-Kawecka
1,2,9
, Clare Stirzaker
1,2,9
, Kee-Ming Chia
3
, Neil Portman
2,3
, 5
Elyssa Campbell
1
, Qian Du
1,2
, Geraldine Laven-Law
4
, Shalima S. Nair
1
, Aliza Yong
3
, 6
Ashleigh Wilkinson
3
, Samuel Clifton
1
, Heloisa H. Milioli
2,3
, Anthony Schmitt
5
, Emily 7
S. Wong
6,7
, Theresa E. Hickey
4
, Elgene Lim
2,3
, Susan J. Clark
1,2,8
8
9
1
Epigenetics Research Laboratory, Genomics and Epigenetics Theme, The Garvan 10
Institute of Medical Research, Sydney, New South Wales, Australia 11
2
St Vincent’s Clinical School, UNSW Sydney, New South Wales, Australia 12
3
Connie Johnson Breast Cancer Research Laboratory, The Garvan Institute of 13
Medical Research, Sydney, New South Wales, Australia 14
4
Dame Roma Mitchell Cancer Research Laboratories, Adelaide Medical School, 15
University of Adelaide, Adelaide, South Australia, Australia 16
5
Arima Genomics, Inc., San Diego, California, USA 17
6
Victor Chang Cardiac Institute, Sydney, New South Wales, Australia 18
7
School of Biotechnology and Biomolecular Sciences, UNSW Sydney, New South 19
Wales, Australia 20
8
Lead Contact 21
9
These authors contributed equally 22
23
Correspondence 24
s.clark@garvan.org.au (S.J.C.) 25
26
One line summary: Epigenetic therapy suppresses tumour growth in metastatic ER+ 27
breast cancer via DNA hypomethylation-induced rewiring of long-range ER-mediated 28
enhancer-promoter chromatin interactions to activate ER target genes involved in 29
repression of proliferation and the cell cycle. 30
31
Keywords 32
3D genome architecture; DNA methylation; Decitabine, Hi-C; epigenetic therapy; 33
ER+ breast cancer; endocrine-resistance; transcription factors; enhancers 34
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 22, 2021. ; https://doi.org/10.1101/2021.06.21.449340doi: bioRxiv preprint

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2
Abstract 35
Three-dimensional (3D) epigenome remodelling is emerging as an important 36
mechanism of gene deregulation in cancer. However, its potential as a target to 37
overcome cancer therapy resistance remains largely unaddressed. 38
Here we show that treatment of endocrine-resistant estrogen receptor positive (ER+) 39
breast cancer with an FDA-approved epigenetic therapy Decitabine (5-Aza-mC), 40
results in genome-wide DNA hypomethylation and suppression of tumour growth in 41
preclinical metastatic ER+ breast tumour xenograft models. Systematic integration of 42
matched chromatin conformation capture (Hi-C), Promoter Capture Hi-C, RNA-seq 43
and ER ChIP-seq data revealed widespread effects on epigenome deregulation, 44
including de-compaction of higher order chromatin structure and loss of topologically 45
associating domains (TAD) boundary insulation. Key enhancer ER binding sites were 46
demethylated and re-activated after Decitabine treatment, resulting in new ER-47
mediated enhancer-promoter interactions and concordant activation of tumour-48
suppressive gene pathways. Importantly, we show that the activated ER target genes 49
were also predictive of good outcome in multiple ER+ breast cancer clinical cohorts. 50
Together our study reveals a previously undescribed mechanism of Decitabine in re-51
wiring DNA methylation-dependent 3D genome architecture resulting in suppression 52
of tumour growth, and highlights the potential of epigenetic therapy in targeting ER+ 53
endocrine-resistant breast cancer. 54
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 22, 2021. ; https://doi.org/10.1101/2021.06.21.449340doi: bioRxiv preprint

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Main 55
Around 70% of breast cancers are driven by the Estrogen Receptor-alpha (ER). ER is 56
a critical ligand-activated transcription factor that controls breast cancer cell 57
proliferation and tumour growth upon exposure to estrogenic hormones
1
. Drugs that 58
target ER pathways are highly effective in the treatment of ER+ breast cancer
2,3
, 59
however de novo or acquired resistance to these agents (endocrine resistance) affects 60
a large proportion (>30%) of patients and is the major cause of breast cancer 61
mortality. Therefore, it is critical to understand mechanisms associated with endocrine 62
resistance to provide new therapeutic interventions that prevent or overcome the 63
resistant phenotype for ER+ breast cancer patients. 64
65
Genome-wide ER chromatin binding patterns, called cistromes, are associated with 66
ER-mediated transcriptional activity in endocrine-sensitive and endocrine-resistant 67
breast cancer contexts
1
. ER chromatin binding is primarily located at distal regulatory 68
elements and putative enhancer regions
4-8
. Previous studies carried out in endocrine-69
resistant cell lines have demonstrated that differential ER binding is a key mechanism 70
that drives clinical response to endocrine therapies
4,9
and importantly, this endocrine 71
resistance can be characterised by epigenetic reprogramming of enhancer elements 72
occupied by ER in the endocrine sensitive state
10
. Epigenetic reprogramming involves 73
altered DNA methylation of enhancers that regulate ER-responsive genes and 74
represents a complementary mechanism that may drive primary and acquired 75
endocrine resistance. Enhancer hypermethylation associated with endocrine resistance 76
occludes ER recruitment, which results in transcriptional reprogramming of ER 77
signalling and estrogen independence
10
. 78
79
Gene expression is highly regulated by (3D) genome architecture, from the local level 80
of chromatin compaction to the higher level organisation of topological associated 81
domains (TADs) and chromosome compartments
11
. Alterations to the 3D genome 82
architecture have been described in a number of different cancers, including prostate 83
cancer
12,13
, breast cancer
14-18
, gliomas
19
, and several hematologic cancers
20,21
. 84
Although cancer cells maintain the general pattern of 3D genome folding, distinctive 85
structural changes have been described in cancer genomes at all of levels of 3D 86
organisation
22
. We and others recently reported that 3D genome structure is also 87
disrupted in endocrine-resistant ER+ breast cancer cells
17,18,23
, notably through long-88
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 22, 2021. ; https://doi.org/10.1101/2021.06.21.449340doi: bioRxiv preprint

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range chromatin changes at ER-enhancer binding sites that are DNA hypermethylated 89
in resistant cells
17
. These key findings suggest that DNA hypermethylation promotes 90
both reprogramming of the ER cistrome and 3D genome disorganisation, leading to 91
altered ER transcriptional activity in ER+ endocrine resistant breast cancer. 92
93
DNA demethylating agents such as Decitabine (5-aza-2’-deoxycytidine, DAC) have 94
emerged as a promising therapeutic strategy for treating various cancers
24
. Decitabine 95
is approved by many international regulatory agencies, including the US FDA and the 96
European Commission (EC), for treating haematological cancers
24
. In solid cancers 97
(including colorectal and ovarian cancer), Decitabine has been shown to demethylate 98
regulatory regions that result in re-activation of tumour suppressor genes
25,26
. 99
Additionally, treatment with DNA de-methylating agents has been shown to stimulate 100
immune response pathways in cancer cells through increased transcription of DNA 101
repeat elements, which induces a viral mimicry response
27-29
. However, the direct 102
effect of these epigenetic drugs on the tumour cells, including epigenome and 3D 103
genome structure, remains largely unexplored, especially in clinically relevant 104
patient-derived model systems or clinical samples. 105
106
To test the therapeutic efficacy of epigenetic therapy with Decitabine in endocrine-107
resistant ER+ patient-derived xenograft (PDX) models we assessed the molecular 108
consequences of treatment on DNA methylation, 3D genome architecture and 109
transcriptional pathways. Our data revealed that Decitabine treatment caused an 110
inhibition of endocrine-resistant tumour growth due to 3D epigenome remodelling 111
and re-activation of ER-driven tumour-suppressive gene pathways, highlighting the 112
potential of epigenetic therapy in treatment of ER+ endocrine-resistant breast cancer. 113
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 22, 2021. ; https://doi.org/10.1101/2021.06.21.449340doi: bioRxiv preprint

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Results 114
115
Low-dose Decitabine inhibits tumour growth and decreases cell proliferation 116
To study the efficacy of epigenetic therapy in the context of endocrine-resistant ER+ 117
breast cancer and to establish its impact on the 3D genome and epigenome, we used 118
patient-derived xenograft (PDX) models (Gar15-13, HCI-005) (Fig. 1a). Gar15-13 119
and HCI-005 PDXs were derived from the metastases of two ER+ patients who had 120
disease progression following one or more lines of endocrine therapy and have been 121
used for several pre-clinical studies
30-34
. Using a low, well-tolerated and non-122
cytotoxic dose (0.5mg/kg; Extended Data Fig. 1a) of Decitabine, we first interrogated 123
the anti-cancer effect of epigenetic therapy on tumour growth. Following tumour 124
implantation and initial period of growth (to a volume of 150-200 mm
3
), mice were 125
randomized to twice-weekly injections of PBS (Vehicle) or 0.5mg/kg Decitabine. 126
Treatment continued with twice-weekly measurement of tumour volume for 35 days 127
or until tumour volume exceeded 1000 mm
3
. At endpoint mice were sacrificed and 128
tumour material collected for analysis. In both Gar15-13 and HCI-005 PDX models, 129
Decitabine treatment elicited a strong growth inhibitory response (Fig. 1b, c) and a 130
significant reduction in the proliferative index as measured by Ki-67 positivity at 131
endpoint (Fig. 1d). 132
133
Decitabine induces widespread DNA hypomethylation 134
To directly identify the epigenetic reprogramming events induced by Decitabine 135
treatment, we first analysed genome-wide DNA methylation data generated using 136
Infinium EPIC Methylation arrays. EPIC arrays were performed on four biological 137
replicates of Vehicle and Decitabine-treated PDX tumours at end-point. All 138
Decitabine-treated tumours exhibited genome-wide DNA methylation loss (Fig. 1e, f 139
and Extended Data Fig. 1b, c, P value < 0.0001, two-tailed Mann-Whitney test), with 140
Gar15-13 showing more hypomethylation compared to HCI-005 (Fig. 1e and 141
Extended Data Fig. 1d) (average 14.55% and 8.74%, respectively). To quantify the 142
extent of DNA methylation loss, we identified differentially methylated probes 143
(DMPs) and differentially methylated regions (DMRs) between Vehicle and 144
Decitabine-treated tumours (Supplementary Table 1) and found that in Gar15-13, 145
Decitabine treatment resulted in 279,908 hypomethylated DMPs, which were 146
encompassed by 71,677 DMRs (total size >65Mb, mean size = 898.8, SD = 918.4). 147
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 22, 2021. ; https://doi.org/10.1101/2021.06.21.449340doi: bioRxiv preprint

Frequently asked questions

Q1. What is the effect of the ER binding on the MYO3B promoter?

In Decitabine-treated tumours, the MYO3B promoter displays increased number of interactions with an enhancer, which gains ER binding with Decitabine treatment. 

Q2. What is the ER binding site of the SPATA18 gene?

In Decitabine-treated tumours, the SPATA18 promoter displays an increased number of interactions with an upstream enhancer region, which gains ER binding with Decitabine treatment, concomitant with loss of DNA methylation. 

Q3. What is the author/funder of the study?

(e) Proposed model of tumour growth suppression induced by epigenetic therapy with Decitabine via DNA hypomethylation and subsequent re-wiring of ER-mediated enhancer-promoter interactions resulting in activation of specific ER target genes.(which was not certified by peer review) is the author/funder. 

Q4. What is the ER binding of the SPATA18 gene?

In Decitabine-treated tumours, the KRT8 promoter displays an increased number of interactions with multiple enhancers, one of which gains ER binding with Decitabine treatment. 

Q5. What is the author/funder of this preprint?

In sula tion scor eP < 0.0001D ec1D ec2D ec3 Ve h1 Ve h2 Ve h4S ize3Mb2Mb1Mb0Mbh ijAv erag eIn sula tion Sco re-10Kb 10Kb406080Differential TADs0bpVehicle DecitabineAv erag eIn sula tion Sco re4060800bp-10Kb 10KbVehicle DecitabineCommon TADsklchr31.00.0-1.086.00 Mb 86.50 Mb 87.00 Mb 87.50 Mb 88.00 MbVehicle DecitabineIn sula tion scor echr41.5 1.0 0.5 0.0 -0.5 -1.0 -1.574.00 Mb 74.50 Mb 75.00 Mb 75.50 Mb 76.00 MbIn sula tion scor eVehicle Decitabine(which was not certified by peer review) is the author/funder. 

Q6. What is the significance of the mYO3B gene in ER+ breast cancer?

The relative expression of the MYO3B gene was significantly upregulated in Decitabine-treated tumours (two-tailed t-test P < 0.05 derived from four replicates) and associated with good outcome in ER+ breast cancer patients in the METABRIC cohort. 

Q7. What is the ER survival plot of the SPATA18 gene?

(c) Kaplan–Meier survival plot showing the ability of SPATA18 gene to stratify ER+ breast cancer patients in the METABRIC cohort into good and poor outcome groups. 

Q8. What is the significance of the PAM50 classification in the high and low gene signature-expressing?

Distribution of the PAM50 classification in the high and low gene signature-expressing patients in the ER+ breast cancer(b) Kaplan–Meier survival plot showing ability of the gene signature identified in their study (n = 17 genes) derived from Decitabine-induced ER-mediated enhancerpromoter interactions to stratify ER+ breast cancer patients in the TCGA cohort into good and poor outcome groups. 

Q9. What is the author/funder of this paper?

(k) Snapshot of region on chromosome 3, showing insulation score calculated in Vehicle and Decitabine-treated tumour Hi-C matrixes, demonstrating loss of TAD(which was not certified by peer review) is the author/funder.