RUNX inhibitor suppresses graft‐versus‐host disease through targeting RUNX‐NFATC2 axis

Abstract Patients with refractory graft‐versus‐host disease (GVHD) have a dismal prognosis. Therefore, novel therapeutic targets are still needed to be identified. Runt‐related transcriptional factor (RUNX) family transcription factors are essential transcription factors that mediate the essential roles in effector T cells. However, whether RUNX targeting can suppress, and GVHD is yet unknown. Here, we showed that RUNX family members have a redundant role in directly transactivating NFATC2 expression in T cells. We also found that our novel RUNX inhibitor, Chb‐M’, which is the inhibitor that switches off the entire RUNX family by alkylating agent–conjugated pyrrole‐imidazole (PI) polyamides, inhibited T‐cell receptor mediated T cell proliferation and allogenic T cell response. These were designed to specifically bind to consensus RUNX‐binding sequences (TGTGGT). Chb‐M’ also suppressed the expression of NFATC2 and pro‐inflammatory cytokine genes in vitro. Using xenogeneic GVHD model, mice injected by Chb‐M’ showed almost no sign of GVHD. Especially, the CD4 T cell was decreased and GVHD‐associated cytokines including tissue necrosis factor‐α and granulocyte‐macrophage colony‐stimulating factor were reduced in the peripheral blood of Chb‐M’ injected mice. Taken together, our data demonstrates that RUNX family transcriptionally upregulates NFATC2 in T cells, and RUNX‐NFATC2 axis can be a novel therapeutic target against GVHD.


Nuclear factor of activated T cells (NFAT) is a family of transcription factors identified in activated T cells and promotes the expression
of multiple cytokines and other regulatory molecules [5]. Among NFAT family proteins, NFATC2 is a positive regulator of T helper type 1 (Th1) inflammation [6] and has been recognized as a therapeutic target for aGVHD [7]. Calcineurin inhibitors, such as tacrolimus and cyclosporin, inhibit T cell activation, expansion, and effector function by binding to calcineurin and subsequently inhibiting NFAT-dependent transcription [8]. More specific NFAT-targeting inhibitors have been developed and validated, but have yet to be analyzed in suitable animal models for potential disease improvement [6,9].
In addition, proinflammatory cytokines exert direct effects on aGVHD target tissues [10]. Although Th2 and Th17 subsets are also involved in pathogenesis; the predominance of Th1 subsets is well established in mediating aGVHD pathogenesis [11]. Interleukin-2 (IL-2), interferon-γ (IFN-γ) and tissue necrosis factor-α (TNF-α) are the critical cytokines produced during Th1 differentiation and are important in aGVHD [12]. Granulocyte-macrophage colony-stimulating factor (GM-CSF also known as CSF2) plays an emerging role across a range of inflammatory diseases [13,14]. Recent studies have suggested that donor-derived GM-CSF is a crucial driver of inflammation and a key factor of determining the severity of the GVHD [15] - [17]. Cytokine management for the prophylaxis and treatment of GVHD appears promising, but further efforts are needed to optimize it [18].
The RUNX family of transcription factors (RUNX1, RUNX2, RUNX3) are essential for diverse functions in mammalian cells and regulate the transcription of target genes by recognizing a core consensus DNA-binding sequence classically referred to as 5′-TGTGGT-3′ [19]. RUNX1 and RUNX3 are widely expressed in T cells, in which they play essential roles in T cell development and the acquisition of T cell effector functions [20], and regulate directly IL2 [21], IFNG [22] and CSF2 [23]. RUNX and NFAT proteins as transcription partners bind in a sequence-specific manner to the promoter and other regulatory elements of target genes and they work together to regulate target genes [24,25]. In addition, it has been reported that RUNX2 activates NFATC2 gene expression in human mesenchymal cells [26], but whether NFATC2 is regulated by RUNX family in T cells is largely unknown.
We have reported that chlorambucil (Chb)-conjugated pyrroleimidazole (PI) polyamides, Chb-M' , as a novel RUNX inhibitor which could specifically recognize and bind to RUNX binding sites inhibit RUNX-mediated gene expression [27,28], but therapeutic strategies targeting the regulation of gene expression in T cells by RUNX inhibitors have not yet been reported. Based on the assumption of the presence of functional redundancy among the RUNX family members [29], we hypothesize targeting whole-RUNX family by Chb-M' can down-regulate NFATC2 and pro-inflammatory cytokine genes of T cells, and reduce GVHD severity.
In this report, we show evidence that inhibition of whole RUNX family inhibits NFATC2 expression, T cell proliferation and cytokine expression. Furthermore, we examine that RUNX-inhibitor, Chb-M' , ameliorates xenogeneic GVHD. Our findings suggest that RUNX inhibition therapy by Chb-M' can be a novel therapeutic strategy toward GVHD.

Cell cultures and reagents
The human Jurkat cell line (clone JE6.1) was obtained from The Euro-  GVHD severity was assessed by a scoring system that incorporates five clinical parameters: weight loss, posture (hunching), activity, fur texture and skin integrity as previously reported [30]. Each parameter received a score of 0 (minimum) to 2 (maximum). Mice that had lost > 20% of their original body weight were euthanized. The final scores of the animals that reached the ethical limit score were kept in the dataset for the remaining time points. Peripheral blood were collected and analyzed by flow cytometry at week 4 after transplantation. To remove red blood cells, blood was lysed in RBC lysing buffer (BD Biosciences) to obtain a cell suspension of mononuclear cells.

Xenogeneic GVHD model
As indicated, mice were euthanized at 7 weeks after transplantation to study the GVHD pathologic findings of recipient lungs and livers.
GVHD pathology scores for recipient liver and lung were assigned in a standard way [31,32].

Study approval
All animal studies were properly conducted in accordance with the

Redundant roles of RUNX family members in NFATC2 expression of T cells
We first investigated whether RUNX family regulates NFATC2 in T cells.
From public chromatin immunoprecipitation sequencing (ChIP-seq) data in activated mouse CD4 T cell, RUNX1 binds to promotor region of IL2, IFNG, TNF and CSF2 within islands of active chromatin mark by H3K4 methylation and H3K27 acetylation ( Figure S1A). Also, RUNX1 binds to NFATC2 promotor region in CD4 T cell of mouse and human, and Jurkat T cell acute lymphoblastic leukemia cell line ( Figure 1A).
Since it has been shown that RUNX family members have redundant functions, next, we looked at the association between the whole RUNX Twenty-four hours after treatment, cells were activated by 50 ng/ml PMA and 1 μM ionomycin. Twelve hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of control vector-transduced cells (n = 3). (D) Restoring NFATC2 expression in RUNX-depleted Jurkat cells reverts RUNX-depletion-mediated inhibition of cytokine expression. Non-RUNX-depleted (shLuc) and RUNX-depleted (shRUNX1_2 and shPanRUNX) Jurkat cells transduced with (NFATC2) or without (Empty) lentivirus expressing NFATC2. Cells were treated with 3 μM doxycycline for 24 hours, then activated by 50 ng/ml PMA and 1 μM ionomycin. Twelve hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of control vector-transduced cells (n = 3). Error bars indicate the mean ± standard error (SE) *p < 0.05, **p < 0.01, compared with the control by two-tailed Student's t-test expression ( Figure 1B). We found a weak but significant correlation between each RUNX family gene and NFATC2 gene expression respectively ( Figure 1C). Although not significant in RUNX2, reporter experiments showed NFAT promoter including RUNX consensus binding site ( Figure S1B) increased its reporter activity by overexpression of each RUNX family (Figures 1D and S2). In addition, shRNA knockdown targeting the common region of RUNX family members (PanRUNX) of Jurkat cells resulted in reduced reporter activity of the NFATC2 promoter ( Figure 1E). These results suggest RUNX family have redundant roles in the expression of NFATC2 in human T cells.

Targeting whole RUNX family reduces the expression of NFATC2 and cytokine genes
To investigate whether RUNX is essential for the expression of  (Figures 2A and 2B). In addition, PanRUNX knockdown reduced TNF the most. In contrast, the knockdown of each single RUNX family members reduced IL2 and CSF2 expression, suggesting that the inhibition of these cytokines by suggesting that RUNX family members are enhancing but dispensable in initiating cytokine gene expression, which is consistent with previously reported results [33]. Taken together, these data collectively indicated that RUNX family member positively regulates Th1-associated cytokine genes and CSF2 expression through both enhancing cytokine genes and directly transactivating NFATC2 expressions, supporting the potential of targeting all RUNX families as a strategy to reduce T cell activation.

RUNX inhibitor Chb-M' suppresses effector T cell response
We have previously reported a potent RUNX inhibitor Chb-M' (Figure S4A) and its efficacy in suppressing RUNX-mediated gene expression [27]. In this study, we examined whether its RUNX inhibiting effect is applicable for suppression of effector T cell response through target- significantly reduced alloreactive T cell proliferation ( Figure 3F). These data collectively indicate that RUNX inhibition by Chb-M' could be a novel therapeutic strategy for suppressing effector T cell response.

DISCUSSION
Here, we demonstrate that inhibition of whole RUNX family suppresses NFATC2 expression in human T cells, inhibits T cell proliferation and cytokine production, and the suppression of xenogeneic-GVHD by RUNX inhibitor, Chb-M' .
The important role that RUNX plays in T cell differentiation has been widely studied, but regulation of NFATC2 expression by RUNX family in human T cells has been poorly studied to date. Although we presented data for public Chip-seq data of only RUNX1 (Figure 1A), RUNX2 and RUNX3 can also bind to the same site because all RUNX family members share the RUNX-binding consensus sequences 5′-TGTGGT-3′ or 5′-TGCGGT-3′. In addition, not only RUNX1, but also RUNX2 and RUNX3 showed a correlation between NFATC2 and their gene expression levels in human CD4 T cells ( Figures 1B and 1C), and reporter experiments showed that RUNX1-3 increases the transcriptional activity of NFATC2, respectively ( Figure 1D Error bars indicate the mean ± standard error (SE). *p < 0.05, **p < 0.01, one-way analysis of variance, followed by the Tukey post hoc test cytokines [45]. A possible reason for the discrepancy between these reports and our results may be that there is compensation by other RUNX families in a single Runx knockout model. Thus, we would like to emphasize that targeting all RUNX family can be effective in reducing suppressing effector T cell activation. We acknowledge that targeting whole RUNX family may also have a negative effect on T cell differentiation in humans, considering that mature thymocyte were virtually absent by reducing core-binding factor β levels in mice, in which the activity of all three Runx proteins should be affected [46]. This is especially important in the context of HSCT, where there is a concern that T-cell differentiation of transplanted donor cells in the thymus may be inhibited, so Chb-M' may be better selected as an additional treatment for SR-GVHD than for GVHD prophylaxis. And, in settings other than HSCT, it could be applied to diseases involving T cell activation such as autoimmune diseases.
We acknowledge that the study has several limitations. First, we only analyzed the effect of Chb-M' on GVHD and not the graft-versusleukemia (GVL) effect. The challenge in the current era is to prevent GVHD while sparing GVL effect. Donor CD8 T cells play a dominant role in mediating the GVL effect via cytotoxic T lymphocyte activity [47]. An essential role of Runx3 in cytotoxic function in activated mature CD8 T cells has been suggested by studies using Runx3deficient mice CD8+ T cells in vitro [48] and in vivo [49]. Thus, the therapeutic effect of GVHD with Chb-M' may also be accompanied by suppression of GVL resulting in the risk of relapse of malignant disease.
However, this may be offset by the antineoplastic effect of Chb-M' depending on the type of malignant disease [27]. Secondly, despite the fact that RUNX1 is an interacting transcriptional partner for FOXP3 [21] and also regulates the expression of FOXP3 in Tregs [50], we did not examine the effect of Chb-M' on Tregs, which are critical mediators of immune tolerance after allogenic HSCT. Although it is possible that Chb-M' may cause suppression of regulatory T cell production as well as conventional T cells, one strategy may be to combine low-dose IL2 treatment with Chb-M' , which has been used in recent years to restore Treg in clinical practice [37]. The effect of Chb-M' on GVL and Tregs will be the subject of further studies.
In conclusion, we have shown that targeting the whole RUNX family suppresses NFATC2 expression and various cytokine gene expressions in T cells. Considering Chb-M' is expected to have therapeutic effects through a mechanism different from other existing GVHD drugs, the first priority is to test whether it is effective as an additional treatment for refractory GVHD in the clinical settings. Further studies are awaited to validate this new strategy against GVHD.