PROGRAM CONTACT:

SUMMARY STATEMENT

( Privileged Communication ) Release Date: 11/09/2015

Principal Investigator 

FAUBION, WILLIAM A MD

Applicant Organization: MAYO CLINIC ROCHESTER

Review Group: GMPB

Application Number: 2 R01 AI089714-06A1

Gastrointestinal Mucosal Pathobiology Study Section

Meeting Date: 10/22/2015 RFA/PA: PA13-302

Council: JAN 2016 PCC: I4A

Requested Start: 07/01/2016 Dual PCC: NLM DUAL

Dual IC(s): DK

Project Title: Inflammatory cascades disrupt Treg function through epigenetic mechanisms

SRG Action: Impact Score: Percentile:

Next Steps: Visit http://grants.nih.gov/grants/next_steps.htm

Human Subjects: 10-No human subjects involved

Animal Subjects: 30-Vertebrate animals involved - no SRG concerns noted

Project 

Year

6

7

8

9

10

TOTAL

Direct Costs 

Requested

Estimated 

Total Cost

ADMINISTRATIVE BUDGET NOTE: The budget shown is the requested budget and has not been 

adjusted to reflect any recommendations made by reviewers. If an award is planned, the costs will be 

calculated by Institute grants management staff based on the recommendations outlined below in the 

COMMITTEE BUDGET RECOMMENDATIONS section.

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2 R01 AI089714-06A1 2 GMPB 

FAUBION, W 

2R01AI089714-06A1 Faubion, William

RESUME AND SUMMARY OF DISCUSSION: This outstanding application from a well-established 

physician scientist is aimed at testing the hypothesis that histone methyltransferase EZH2, through 

epigenetic modification of Foxp3, plays a critical role in the homeostasis of Treg cells and that 

disruption of EZH2 function contributes to inflammatory bowel disease. During the previous period of 

funding, the principal investigator has been highly productive with important findings on EZH2-mediated 

transcriptional repression in Tregs. During a focused discussion, the reviewers noted numerous 

strengths of the application including the highly significant topic of research with clinical relevance and 

potential high impact on broad areas of autoimmune disease, the highly regarded and productive 

investigator, the innovative concepts regarding the epigenetic modifications of FoxP3 and cell 

autonomous mechanism of IL-6 in disrupting Treg function, the strong supportive preliminary data, the 

logically designed experiments with clear outcomes, as well as the exceptional research environment. 

Only minor concerns were noted and this application was viewed with high uniform enthusiasm. 

DESCRIPTION (provided by applicant): The transcription factor FOXP3 is critical to the regulation of 

numerous debilitating human immune-mediated diseases. Very recently, the essential role for the 

histone methyltransferase (HMT) EZH2 in the epigenetic regulation and function of FOXP3 has been 

described. Inflammatory pathways modify EZH2 activity, and inflammatory signaling impairs Treg 

function in vivo and in vitro. The biological impact of the FOXP3-EZH2 pathway to IBD is unknown. Our 

long-term goal is to dissect epigenetic mechanisms regulating Treg cellular differentiation and function, 

particularly within the setting of GI inflammatory diseases. These discoveries will facilitate design of 

human cell therapy trials for IBD. The objective of this grant is to characterize the role for EZH2 in Treg 

suppressive function. The central hypothesis is that EZH2 plays a critical role in the homeostasis of 

Treg cells, and the disruption of EZH2 function by inflammatory signaling pathways contributes to IBD. 

Our rationale is that identification of the mechanism(s) to restore Treg suppressive function in the 

setting of intestinal inflammation will offer new therapeutic opportunities. Our specific aims will test the 

following hypotheses: (Aim1) Repression of immunoregulatory gene networks by FOXP3 requires the 

formation of a complex between this transcription factor and EZH2; (Aim 2) Inflammatory stimuli, such 

as IL6 lead to EZH2 phosphorylation and thereby disrupt the enzymatic activity of this epigenomic 

regulator; (Aim 3) Inhibition of the IL6 to EZH2 signaling pathway permits sustained Treg suppressive 

function in the setting of intestinal inflammation. Upon conclusion, we will understand the role for EZH2 

in Treg loss of function in the setting of active inflammation. This contribution is significant since it will 

establish that several pathways targeted by available therapies (ie IL1β, IL6, TNFα) have the potential 

to regulate EZH2 HMT activity through post- translational modifications. Furthermore, current Treg cell 

therapy trials, while promising have not addressed the key issue of in vivo inflammation-induced 

disruption of Treg function. The proposed research is innovative because we investigate the effect of 

inflammatory signaling pathways on epigenetic complexes in Treg cells, a heretofore-unexamined 

process. Insight into epigenetic mechanisms is impactful as T cell progenitor cells inherit the parent 

transcriptional profile and unlike genetic change, they are modifiable by currently available therapy. 

PUBLIC HEALTH RELEVANCE: The proposed research is relevant to the public health because IBD, 

increasing in prevalence, represents a major national cost measured by both patient suffering and 

economic burden; and despite significant advances in care, clinical trial data demonstrate remission 

rates at best of 40%. Upon conclusion, we will understand the role for EZH2 in Treg loss of function in 

the setting of active inflammation, and this discovery will stimulate the opening of a new avenue in 

therapeutics directed at stimulation of autologous Treg cells to function within the inflammatory milieu. 

CRITIQUE 1 
2 R01 AI089714-06A1 3 GMPB 

FAUBION, W 

Significance: 2 

Investigator(s): 3 

Innovation: 3 

Approach: 3 

Environment: 2 

Overall Impact: Dr. Faubion is an excellent physician-scientist who is an up and coming leader in the 

area of Treg biology related to GI diseases. He has published a number of very good manuscripts in 

the first funding period investigating the role of transcription factors and modulation of chromatin 

modifying complexes in Treg biology. In this application, he proposes to study the exciting area of 

epigenetic modulation of Treg suppressive function through the EZH2 signaling pathway. The area of 

investigation is considered highly significant. Globally, while studied by other leading investigators, 

pursuing regulation of epigenetic modifications of FoxP3 and Treg biology as a therapeutic approach 

for IBD is considered innovative. Enthusiasm for this application was somewhat dampened by the 

failure by the applicant to integrate and discuss the findings of three high profile recent papers that are 

directly related to this application (and suggest alternative mechanisms). In addition, the studies 

assessing the role of IL-6 related biology on the regulation of EZH2-mediated signaling would be 

strengthened by more preliminary data. 

1. Significance: 

Strengths 

Deciphering the role of epigenetic regulation of FoxP3 and Tregs has clear relevance to 

immune-mediated disease. Dr. Faubion’s group made the initial discovery, confirmed by others 

in high impact journals, of a role for the histone methyl transferase EZH2 in the regulation of 

FoxP3. 

Inflammatory processes that potentially paralyze the HMT activity of EZH2 has relevance to 

Treg-targeted therapies. 

2. Investigator(s): 

Strengths

Dr. Faubion is an excellent physician scientist. He is an Associate Professor of Pediatrics, 

Medicine and Immunology at the Mayo Clinic. 

He has published four senior authored manuscripts in the last five years in very good peer 

review journals (1 CMGH, 2 AJP, and 1 JBC) in the area of KLF regulation of Treg and 

lymphocyte biology. 

He is currently funded by the . 

3. Innovation: 

Strengths 

Directly investigating the role of epigenetic modifications on FoxP3 and Treg-mediated 

suppression and the role that inflammatory cytokines have in this process is considered 

innovative. As such, it is not surprising that this area has been extensively studied by a number 

of leading investigators (Bluestone, Rudensky, Laurence). 

4. Approach: 

Strengths 

The generation of conditionally targeted EZH2 mice (in FoxP3 positive cells) - FOXP3ΔEZH2 - that 

results in a lethal inflammatory disorder supports the hypothesis of a critical role of EZH2 in the 

epigenetic regulation of Tregs. 
2 R01 AI089714-06A1 4 GMPB 

FAUBION, W 

Direct demonstration of interaction between EZH2 and FoxP3 support studies in Aim 1 which 

will investigate the critical domains required for functional activity. These experiments will yield 

clear results. 

Direct translation of these findings to EZH2 gene targets of relevance in IBD (RORA and IRF4) 

– both upregulated in CD – in reporter assays and ChIP assays with WT FOXP3/EZH2 and 

domain mutants are well outlined and focused (albeit perhaps too focused). 

Studies in Aim 2 to determine the role of the role of IL-6 on EZH-mediated suppression are likely 

to yield definitive and informative data. Similarly, experiments addressing the role of an EZH 

phosphomimetic Y641mutant and the non nonphosphorylatable Y641F mutant in vitro (Aim 2) 

and in vivo (Aim 3) are likely to yield informative data. 

Weaknesses 

Defective function of Tregs in vitro contrasts with recent publications by Bluestone et al –

suggesting normal in vitro function. These differences are not discussed and should since they 

may point different mechanistic functions. Indeed these differences are relevant to the in vitro 

suppression assays outlined in several sub aims. 

In addition to the unpublished worked presented in this proposal, data pointing to the role of 

EZH2/FoxP3 interactions in regulating colitis has been recently published by Laurence et al. 

These data due support the use of specific constructs to determine the precise interactions 

necessary to mediate EZH2-mediated suppression. However, the statement that “these exciting 

experiments will be the first to test a modifiable epigenetic mechanism in the treatment” of IBD 

is exaggerated. 

Rudensky et al has suggested that inflammatory signals induce the EZH2-Foxp3 mediated 

suppressive program. These data are counter to the hypothesis proposed and necessitates 

discussion. 

In this regard, the potential role of IL-6-driven and JAK2-mediated phosphorylation of EZH2 

leading to inhibition of EZH2-dependent suppression while exciting would be strengthened 

markedly by preliminary data showing that IL-6 fails to suppress Treg function in the absence of 

EZH2. 

Studies investigating the role of blocking IL-6R in Tregs as a mechanism for treating colitis while 

potentially related to EZH2 may have many other possible mechanisms. The studies outlined, 

while potentially clinically relevant, do not support a direct role of IL-6R-activation in inhibiting 

EZH2 mediated suppression. Nonetheless, as noted above expression of the EZH 

phosphomimetic Y641mutant and the non nonphosphorylatable Y641F in Tregs will indirectly 

support a role of IL-6R activation in this process. 

5. Environment: 

Strengths 

Outstanding environment at the Mayo Clinic. 

Protections for Human Subjects: Not Applicable (No Human Subjects) 

Vertebrate Animals: YES, all five points addressed 

Biohazards: Acceptable 

Resubmission: Overall the applicant responded to the initial critiques. The applicant has added three 

new senior authored publications to his portfolio. He adequately addressed the extension of his original 

research project on KLF to study of EZH2. 

Renewal: The applicant has published 10 peer review papers related to his initial project in very good 

(but not top) journals. 
2 R01 AI089714-06A1 5 GMPB 

FAUBION, W 

Budget and Period of Support: Recommend as Requested

CRITIQUE 2 

Significance: 1 

Investigator(s): 1 

Innovation: 2 

Approach: 2 

Environment: 1 

Overall Impact: Goal of this revised competitive renewal R01 application is to further define the roles 

and mechanisms of FOXP3 and Treg cells in the pathogenesis of IBD, with particularly focusing on an 

epigenetic regulator histone methyltransferase (EZH2). Experiments outlined in the current proposal 

are natural extension of the PI previous works, but they are to further explore a new area by testing an 

interesting and novel hypothesis with considerable amounts of supporting preliminary results. The 

proposal is based on a strong rationale, well-prepared, and has a great potential impact in clinically 

important area of IBD. The productivity of the previous funding period is very good. The PI is a leader 

in this area and continuously successes in basic and translational research. All questions and 

concerns raised by previous study section have been nicely addressed by the PI, and some new 

preliminary results are also provided. Although there are still some minor concerns with the revised 

application, they don’t affect overall enthusiasm to this proposal, based on PI track record and 

experience. The impact of this project is pretty high and it would provide crucial information about our 

understanding of EZH2 in IBD. 

1. Significance: 

Strengths

Studies proposed in the application address key aspects of FOXP3 and Treg cells in the 

immune-response to inflammation in the gut mucosa and have great potential impact on the 

important area of IBD.

To define biological role of EZH2 and its regulatory function on FOXP3 and Treg cells is novel 

and of significant. 

Many of the techniques proposed as well as recently generated new conditional knockout mice 

models are also novel.

Experiments investigating EZH2 phosphorylation events during inflammation would provide 

significant information regarding gene regulatory cascade and it is also a potential new target of 

immunotherapy for patients with IBD. 

Weaknesses

Several previous studies have shown that IL-6 mediated disruption of Treg function is not totally 

dependent on EZH2. 

2. Investigator(s): 

Strengths 

The PI is an outstanding physician scientist with an excellent track record and particular 

expertise for the proposed experiments. 

The PI and this creative group are able to carry out all experiments outlined in the current 

application. 
2 R01 AI089714-06A1 6 GMPB 

FAUBION, W 

Weaknesses 

The productivity of previous funding period is moderate. 

3. Innovation: 

Strengths 

Studies defining the mechanisms by which EZH2 modulates Treg cells and FOXP3 transcription 

factor in the setting of intestinal inflammation by using various genetically modified mouse 

models and state-of-the-art techniques are highly innovative. 

Weaknesses

Mechanisms underlying IL-6 mediated disruption of Treg cell function have been previously 

reported by many labs. 

4. Approach: 

Strengths 

Experiments are nicely and logically designed with appropriate controls proposed, and the 

protocol is easy to be read and followed. 

All necessary animal models and in vitro systems, functional and molecular biology assays for 

proposed studies are available and have been used on daily base for many years in the PI’s lab. 

Alternative plan is also provided in the proposal. 

There are considerable amounts of preliminary results with the revised application, which 

support the hypothesis and verify the feasibility of methods. 

Weaknesses 

The novelty of some experiments in Aim #2 needs to be improved.

5. Environment: 

Strengths 

The environment is excellent at Mayo clinic. 

The lab and available facility of the PI are suitable for completing experiments proposed in the 

current application. 

Protections for Human Subjects: Not Applicable (No Human Subjects) 

Vertebrate Animals: YES, all five points addressed 

Biohazards: Acceptable 

Resubmission: All questions are addressed nicely by the PI, and some new preliminary results are 

also provided in the revised application. The proposal is improved significantly in general. Some 

technical concerns are minor and would be solved easily by the PI. 

Resource Sharing Plans: Acceptable 

Budget and Period of Support: Recommend as Requested 

CRITIQUE 3 

Significance: 1 

Investigator(s): 1 

Innovation: 1 
2 R01 AI089714-06A1 7 GMPB 

FAUBION, W 

Approach: 1 

Environment: 1 

Overall Impact: This A1 revised application proposes to investigate how inflammatory stimulus such as 

IL-6 disrupts EZH2-mediated transcriptional repression of FOXP3 target genes in IBD. In their last 

funding cycle, this group discovered that EZH2-mediated transcriptional repression is essential for Treg 

suppressor function. The previous review of this grant acknowledged the outstanding expertise of this 

investigator. The significance of the proposal was deemed quite strong, with possible translation into 

clinical setting and immunotherapy, and broad applicability in inflammatory diseases. The grant was 

considered high on innovation. Previous concerns were raised regarding the approach. Here, the 

investigator responds positively and is able to adequately address most of the concerns raised during 

the earlier review. This includes the addition of significant new data that supports their hypothesis and 

demonstration of feasibility. Additionally, some concerns were noted about publication record, which 

has been satisfactorily addressed by the investigator. Therefore, the overall impact of this outstanding 

proposal is now considered to be high. 

1. Significance: 

Strengths 

Concerns were noted in the last review regarding sufficiency of EZH2 in regulating Treg 

function. New data is presented demonstrating FOXP3DEZH2 shows enhanced susceptibility to 

DSS-induced colitis. FOXP3DEZH2 lymphocytes also show increased IFNγ and IL-17, and 

reduced IL10 production - perhaps by inhibition of a co-activator of IL-10 or indirectly in 

response to IFNγ and IL-17. Additionally, co-transfer of FoxP3+ cells following inducible deletion 

of EZH2 did not confer protection in a T cell-transfer model of colitis.

Published accounts of IL-6 mediated disruption of Treg function exists. Notwithstanding, this 

project appears unique in proposing a cell autonomous mechanism of I-L6 action on FOXP3+ T 

cells. 

2. Investigator(s): 

Strengths

While the publication record of the investigator was lauded, there were some concerns about 

paper with senior authorship and impact factor of journals published in. The concern was 

particularly raised in the backdrop of what was considered a change in direction (EZH2-

focussed as opposed to KLF10-focussed). The investigator now lists 10 papers related to the 

project, including 6 as senior author. Additional 6 papers are listed outside of the focus of the 

prior grant. This raises the confidence in his ability to produce results. Additionally, the 

investigator now specifically points out a flow of logic linking KLF10 to EZH2. Therefore, it is felt 

that the investigator addressed this concern adequately.

3. Innovation: 

Strengths

The proposal centers on a novel, cell autonomous mechanism of IL-6 action in disrupting Treg 

function. 

4. Approach: 

Strengths 

Previously, a number of concerns were noted in this section. The investigator responded 

positively and now provide substantial amount of new data to support feasibility. 
2 R01 AI089714-06A1 8 GMPB 

FAUBION, W 

It was enquired whether FOXP3 EZH2 mice shows spontaneous colitis. The investigator now 

demonstrates, albeit not spontaneous colitis, enhanced susceptibility to 1% DSS. This can be 

considered supportive of the investigators hypothesis. The occurrence of spontaneous colitis is 

somewhat rare such as in in genetic ablation of IL10, and other models of deletion of important 

anti-inflammatory molecules such as TAM RTKs show induced, but not spontaneous, colitis. In 

humans, while the etiology remains unknown, the incidence of colitis may not be spontaneous 

and may depend upon environmental factors. 

It was suggested that involvement of STAT3 be considered. The investigator now refers to 

EZH2-mediated methylation of STAT3 in glioblastoma in alternative approaches and proposes 

to isolate EZH2 complex after IL6 treatment to identify binding partners and substrates. 

Feasibility of neonatal cell transfer experiments was questioned. The investigator jettisoned 

those experiments in the revised proposal, thus alleviating this specific concern. 

Cytokine profiling of colonic supernatants is included in Aim 1.3 as was suggested by Previous 

Reviewer 1. 

Substantial amount of new data is provided in support of Aim 2. Phosphorylation site was 

identified and phosphomimetic and nonphosphorylatable mutants are described. 

Concerns about progress in the previous funding period along with lack of some proposed 

studies viz. using KLF10flox/flox raised doubts about the investigators ability. The new preliminary 

data provided by the investigator, including new tools such as inducible deletion of EZH2 and 

phosphosite studies mostly alleviates the concerns. 

In response to a question, the investigator includes new data on the expression of 

immunoregulatory molecules and IFNγ, IL17 and IL10 in FOXP3 EZH2 Tregs. 

Concerns about high levels of cytokine in KO and spontaneous inflammatory pathology can be 

addressed by inducible ablation. 

The investigator added new data on T cell-transfer model. 

The investigator was amenable to the recommendation to improve potential pitfalls and 

alternative approaches for Aim 3. 

Weaknesses 

EZH2 action in other lymphocytes was indicated as an area of concern. This point could have 

been addressed better. 

The downregulation of IL10 following ablation of a repressive histone methyltransferase function 

remains a bit of conundrum not addressed by the investigator. 

5. Environment: 

Strengths 

Mayo clinic is an outstanding institution and provides the right environment to perform the 

proposed work. 

Protections for Human Subjects: Not Applicable (No Human Subjects) 

Vertebrate Animals: YES, all five points addressed 

Biohazards: Not Applicable (No Biohazards) 

Resubmission: The applicant has adequately addressed the previous comments. 

Resource Sharing Plans: Acceptable 

Budget and Period of Support: Recommend as Requested 
2 R01 AI089714-06A1 9 GMPB 

FAUBION, W 

THE FOLLOWING SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO 

SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE, OR REVIEWERS' 

WRITTEN CRITIQUES, ON THE FOLLOWING ISSUES: 

VERTEBRATE ANIMAL (Resume): ACCEPTABLE 

COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested. 

NIH has modified its policy regarding the receipt of resubmissions (amended applications). 

See Guide Notice NOT-OD-14-074 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD14-074.html. The impact/priority score is calculated after discussion of an application by 

averaging the overall scores (1-9) given by all voting reviewers on the committee and 

multiplying by 10. The criterion scores are submitted prior to the meeting by the individual 

reviewers assigned to an application, and are not discussed specifically at the review meeting 

or calculated into the overall impact score. Some applications also receive a percentile 

ranking. For details on the review process, see 

http://grants.nih.gov/grants/peer_review_process.htm#scoring.