Next generation treatment of acute graft-versus-host disease.

Accepted Article Preview: Published ahead of advance online publication Next Generation Treatment of Acute Graft-versus-Host Disease John Magenau, Pavan Reddy

Cite this article as: John Magenau, Pavan Reddy, Next Generation Treatment of Acute Graft-versus-Host Disease, Leukemia accepted article preview 18 June 2014; doi: 10.1038/leu.2014.195. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Received 17 April 2014; revised 23 May 2014; accepted 4 June 2014; Accepted article preview online 18 June 2014

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2014 Macmillan Publishers Limited. All rights reserved.

LEUKEMIA – REVIEW

Title: Next Generation Treatment of Acute Graft-versus-Host Disease Running Title: Next Gen GVHD Treatment Keywords: GVHD, Allogeneic, Transplantation

Authors: John Magenau, M.D.* Clinical Assistant Professor [email protected] Pavan Reddy, M.D. * Professor of Internal Medicine [email protected] Blood and Marrow Transplant Program Division of Hematology / Oncology Department of Internal Medicine University of Michigan Medical School 3312 Cancer Center 1500 E. Medical Center Drive Ann Arbor, MI 48109-5932 (Ph) (734) 936-8785 (fax) (734) 232-4484

*Address correspondence The authors report no conflicts of interest.

Word count: 4390 Figures: 3 Tables: 1 References: 86

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ABSTRACT Despite rapid increase in the utilization of allogeneic hematopoietic stem cell transplantation (HCT), non-relapse mortality and sequela from acute graft-versus-host disease (GVHD) remain principle barriers. GVHD involves complex interactions between innate and adaptive immunity, culminating in tissue damage by inflammatory mediators and cellular effectors. Recently, our understanding of the molecular intricacies of GVHD has grown tremendously. New insights into the roles played by novel cytokines, chemokines, intracellular signaling pathways, epigenetics and post translational modifications of proteins in GVHD biology provide numerous targets that might be therapeutically exploited. This review highlights recent advances and identifies opportunities for reshaping contemporary GVHD therapeutics.

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INTRODUCTION Allogeneic hematopoietic stem cell transplantation (HCT) possesses curative potential for many aggressive hematologic malignancies, but its success remains limited by acute graft-versus-host disease (GVHD)(1, 2). Clinically significant GVHD (grades II-IV) affects up to half of patients receiving human leukocyte antigen (HLA) identical allografts, resulting in significant morbid sequela from organ damage and impaired immunity (3, 4). Because the success of HCT depends on simultaneous graft-versustumor (GVT) effects, broad-based immunosuppressive strategies are less attractive. Moreover, relapse accounts for a significant proportion of treatment failures after HCT (2), thus strategies for GVHD prevention with minimal impact on GVT would be ideal. This review focuses on recent advances in the immunobiology of GVHD and provides examples of selective targeting that may minimize deleterious effects on immunity.

Despite our current state of understanding, Billingham's postulates remain hallmarks of GVHD. The essential components include: (i) the presence of immune competent cells from the donor (ii) the inability of the recipient to reject donor cells and (iii) histocompatibility differences between the donor and recipient (5). Donor T cells are now recognized as occupying a central role in mediating GVHD following interactions with activated host and donor antigen presenting cells (APC)(6). A complex network of cytokines, chemokines, cellular receptors and immune cell subsets then modulate T cell / APC interactions that result in the initiation and maintenance of GVHD (7). Contemporary GVHD prophylaxis at most centers is based on calcineurin inhibitors (CNIs) along with short-course methotrexate (MTX)(8). By interfering with calcium 3

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dependent IL-2 gene activation and de novo purine metabolism, respectively, CNIs and MTX act synergistically to non-selectively inhibit lymphocyte activation and proliferation. Intensifying GVHD prophylaxis by widespread T cell depletion with anti-thymocyte globulin (ATG) prevents GVHD, but without improving survival due to offsetting risks for relapse, infection and graft rejection (4, 9). With standard prophylaxis, approximately 40% of patients receiving HLA matched HCT will develop GVHD requiring high dose corticosteroids (3, 10). However, up to 60% of patients will have an inadequate responses to corticosteroids, which portends a dismal prognosis (11). Considering the increased utilization of HCT, the morbidity and mortality associated with GVHD and the limitations inherent to contemporary therapies, novel approaches are urgently needed. Rationally designed treatments that inhibit deregulated pathways in malignant cells are typically the focus of ‘targeted’ therapy. In GVHD the ‘target’ is elusive, since the goal is to carefully contain an overzealous, but biologically normal immunologic response. Herein we emphasize potential novel approaches that may be selectively interrupted to limit immunological impairment based on recent preclinical insights from animal models addressing the biology of GVHD. While undoubtedly inspired from pioneering work in murine HCT and seminal observations from human trials, this review focuses primarily on opportunities for future clinical translation; as such we will not describe many of the current treatments in the clinic. For conceptual ease, we have organized the diverse approaches into three groups: 1) extracellular mediators and receptors 2) intracellular signal transduction and 3) regulation of transcription / translation (Figure 1). Furthermore, considering the large body of recent work, but in light of the space constraints, we have regrettably been unable to cite all relevant original studies. 4

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Extracellular mediators and receptors Our knowledge of how cell surface receptors regulate immune responses has grown substantially. By coordinating intracellular responses to a multitude of endogenous and exogenous inflammatory signals, immune receptors ultimately initiate signaling cascades instrumental for the initiation and amplification of GVHD (Figure 2). Cytokines and chemokines: Cytokines and chemokines influence T cell differentiation pathways and trafficking to GVHD target organs, but they also mediate direct tissue injurious effects. In the past, blockade of single cytokines, such as TNFα or IL-1, along with CNIs, did not prove effective for clinical GVHD (12, 13). The reasons for this lack of translation are likely multiple but could relate to impaired TNFα or IL-1 antagonism in the presence of CNIs or the heterogeneous design of trials during their clinical development. Nevertheless, considering these experiences and acknowledging the complexity of GVHD, it remains to be seen whether interruption of single cytokines will prove viable in the clinic. For example, blockade of multiple effector pathways that include T cell intrinsic pathways may ultimately be necessary. However, recent insights suggest novel cytokines play multiple divergent roles in GVHD pathobiology with potential salutary effects on tissue repair and GVT, thus renewed interest in targeting these pathways may be worthwhile. Interleukin-6 (IL-6): There is mounting evidence that IL-6 acts as a key inflammatory cytokine in GVHD pathogenesis. IL-6 regulates GVHD by increasing Th1 and Th17 subsets, decreasing regulatory T cells (Tregs) and by direct cytotoxicity (14, 15). Post 5

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HCT IL-6 blockade with IL-6(-/-) mice or with antibody strategies significantly reduced experimental GVHD without impairing GVT. In the clinic, the anti-IL-6 receptor antibody tocilizumab, now FDA approved for rheumatoid arthritis, demonstrated activity when administered to a small series of patients with grade IV GVHD (16). In data reported in abstract form, adding tocilizumab to standard GVHD prophylaxis after HLA matched HCT resulted in exceptionally low grade II-IV acute GVHD rates versus historical controls, without notable worsening of relapse or CMV re-activation (17). Interleukin-21 (IL-21): has also emerged as an important regulator of Th1 and Th17 differentiation while simultaneously inhibiting the production of inducible (iTregs). Administration of IL-21 inhibitors or IL-21 deficient T cells mitigated murine gastrointestinal (GI) GVHD (18). Furthermore, despite a reduction in GI GVHD, IL-21 inhibitors did not attenuate GVT effects (18). IL-21 protein expression was increased in clinical samples from the GI tracts of patients with GVHD and in a human xenograft model prophylactic administration of anti-human IL-21 reduced disease lethality (19). Interleukin-23 (IL-23): has similarly been implicated in experimental GI GVHD. Donor bone marrow deficient in IL-23 reduced GI GVHD compared to animals receiving wildtype grafts. In DCs purified from the colon, levels of IL-23 and its receptor were elevated, but not at other GVHD target sites. Increased IFN-γ CD4+ T cells appear to mediate these inflammatory effects (20). CCR5: Interrupting immune cell trafficking prevents interactions with APCs that initiate GVHD. For example, the chemokine (C-C motif) receptor-5 (CCR5) is upregulated in T lymphocytes upon allogeneic stimulation and directs homing to target tissues (21). This

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is supported by the observation that HCT recipients harboring CCR5 Delta32 mutation (resulting in loss of function) are protected against GVHD (22). Experimentally, CCR5 deficiency has not consistently demonstrated a GVHD protective effect, possibly related to differences in strains and conditioning (23). In a recently completed human study, Maraviroc, a small molecule antagonist for CCR5 trophic HIV, was added to standard GVHD prophylaxis with promising results that are being further studied (24). Attention has also begun to focus on cytokines and intestinal stem cells (ISCs) involved in epithelial repair. ISCs are sensitive to the cytotoxicity of transplant conditioning and GVHD. Likewise, interleukin-22 (IL-22), produced by innate lymphoid and T cells of the intestine can reduce tissue sensitivity to GVHD. These observations are unified by the finding that deficiency of IL-22 promotes loss of ISCs, intestinal damage and greater experimental GVHD (25). Although IL-22 is not available clinically, Wnt agonists such as R-spondin1 (R-Spo1) might similarly protect ISCs and consequently enable repair of intestinal epithelium. Indeed, R-Spo1 inhibits inflammatory cytokines, facilitates intestinal healing and interrupts the amplification of GVHD in murine models (26). Since the intestine is a primary target organ, Wnt agonists might be useful in limiting the tissue injurious effects of conditioning and GVHD. Alpha-1-antitrypsin (AAT): AAT is a serine protease inhibitor produced in the liver and known to inhibit neutrophil elastase, whose deficiency results in emphysema and cirrhosis (27). Human AAT treatment was first shown to promote allograft tolerance in models of islet cell transplantation (28), and subsequent work showed that AAT also induces tolerance in

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the allogeneic setting by inhibiting multiple pro-inflammatory cytokines. In models of MHC disparate and matched GVHD, human AAT administered before and immediately after HCT reduced GVHD, increased Treg to T effector ratios, and did not impair T cell responses (29). Treated animals had significantly reduced levels of multiple proinflammatory cytokines, but increased secretion of the anti-inflammatory cytokine IL-10, suggesting AAT affects experimental GVHD via modulating host DC responses. Similar results have been observed by several groups (30, 31). The mechanism by which AAT attenuates DC responses may involve diminished tissue production of damage associated noninfectious signals (DAMPs) such as heparin sulfate (HS), which is elevated in experimental GVHD (31). In humans, elevated concentrations of AAT have been observed in stool samples from patients with severe GI GVHD (32), and together with calprotectin predicted steroid refractoriness. Given these observations, a pilot clinical trial of exogenous AAT supplementation in steroid-refractory GVHD is underway. Cellular adhesion: The adhesion molecule DNAX accessory molecule 1 (DNAM-1) is constitutively expressed on numerous cells including T cells and DCs (33). The role of DNAM-1 was recently explored in murine GVHD utilizing sub-lethal irradiation (34). DNAM-1 expression was upregulated following activation of T cells and significant expression of its ligands (CD155 and CD112) was observed in GVHD target tissues. Splenocytes deficient in DNAM-1 and prophylactic treatment with anti–DNAM-1 antibody prevented and treated GVHD. The role of DNAM-1 was also investigated in GVT utilizing MHC matched and mismatched models (35). Myeloablation followed by transfer of DNAM-1deficient T cells significantly improved GVHD survival without substantially dampening 8

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GVT. The underlying mechanism appears to be independent of cytokines, but rather from preferential expansion of Tregs. Notch Signaling: Canonical notch signaling plays an essential role in stem cell maintenance and T cell responses. Notch deficient CD4+ T cells or blockade of DL-1 or DL-4 ligands retains proliferative potential, but reduced multiple pro-inflammatory cytokines (36). Diminished GVHD lethality was demonstrated after pan-notch inhibition in murine models of GVHD. Notch signaling in the host hematopoietic APCs could play an important role through Ikaros mediated effects on GVHD and GVT (37). Detection of tissue injury: P2X7 and Nlrp3: The extent of tissue damage from conditioning may alter the severity and the kinetics of GVHD. Classically, tissue damage releases exogenous and endogenous damage / pathogen associated molecules termed DAMPs / PAMPs that are detected by pattern recognition receptors (PRRs). Examples of PRRs include Tolllike Receptors (TLRs) and NOD-like Receptors (NLRs) that result in activation of APCs (7). The purine nucleoside ATP is one DAMP whose release has been implicated as an early danger signal following tissue damage after allogeneic HCT. Elevated levels of ATP are found in peritoneal fluids of humans and mice following GVHD or irradiation. ATP interaction with the purinergic receptor P2X7 on host APCs resulted in increased expression of co-stimulatory molecules CD80/CD86, phosphorylation of STAT1, and production of inflammatory cytokines (38). Interrupting this interaction reduced GVHD mortality but did not mitigate GVT effects in experimental models. 9

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More recently, a multi-protein complex termed the Nlrp3 inflammasome was shown to respond to DAMPs / PAMPs such as uric acid (39). Mice deficient in critical components of Nlrp3 or Asc (adaptor protein for caspace-1 cleavage) demonstrated less severe GVHD. These and other data suggest that DAMPs such as ATP, HS and uric acid may have non-redundant roles in the aggravation of GVHD. Siglecs: Emerging data have demonstrated a role for negative regulators of DAMP responses in controlling the severity of GVHD. Siglecs are a family of sialic acid binding Ig-like lectins which function as counter-regulators to immune activation (40). Following conditioning, Siglec-G is expressed on host APCs and can suppress activating signals from DAMPs (41). Siglec-G-/- animals have increased GVHD, an effect confined to radiosensitive host APCs. In contrast, enhanced Siglec-G signaling with CD24 in wildtype animals was protective. These data suggest that enhancing signaling through siglecs could mitigate GVHD severity. Microbiome: The interplay of the intestinal microbiome and GVHD has been a topic of investigation since the 1970s when van Bekkum observed delayed GVHD after gut decontamination (42). Because 80% of human bacterial flora cannot be cultivated (43), rigorous examination of the relationship between GVHD and microbiota has only been recently analyzed utilizing culture independent rRNA gene sequencing (44, 45). Profound loss of bacterial diversity occurs in mice after GVHD, while absence of microbiota in the donor animals did not influence GVHD in normal recipients (46). Eliminating some species prior to HCT, such as Lactobacillus, correlated with increased GVHD pathology which

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could be reversed by their re-introduction, suggesting that loss of certain commensal bacteria may promote GVHD severity. Likewise, in humans, a shift towards enterococci was observed after conditioning, was further pronounced after antimicrobial usage and was most notable after intestinal GVHD (47). Paneth cells, recently discovered to provide a protective niche for ISCs, interact with the microbiome directly via secretion of the antimicrobial peptide α-defensin (44). Destruction of paneth cells after GVHD reduces α-defensin and drastically alters the intestinal microbiota of mice which may amplify its severity. In human duodenal biopsies taken at GVHD onset, paneth cell loss was associated with disease severity and treatment response (48). Alterations to the microbiome raise new and old questions such as the role of prophylactic antimicrobials, strategies to prevent loss of regenerative cell populations and even re-introduction of commensal flora. The mechanisms and the specific nature of the quality and quantity of the microbiome remain to be better understood. Cellular mediators of tolerance: Ex vivo Treg expansion: Murine models have illustrated that adoptive transfer of CD4+CD25+ Tregs protects animals against lethal GVHD (49, 50). Clinically relevant immunosuppression may require significantly amplified ratios of Tregs to T effectors that are not readily achievable in vivo. Precise control of cellular ratios in the donor cell inoculum could be accomplished by ex vivo expansion and adoptive transfer of Tregs. However, expanding Tregs in culture has historically been hampered by the inability to generate stable populations at high purity. Despite these concerns, adoptive transfer of

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Tregs has now been described in clinical trials utilizing HLA disparate donors at very high risk for GVHD. In haploidentical HCT, donor Tregs were infused four days prior to CD 34+ selected stem cells and low doses of conventional T cells (25 - 50% of Treg). Without prophylactic immunosuppression, only 2 of 26 patients developed ≥ grade II acute GVHD (51). In double-cord HCT, escalating doses of third party expanded Tregs limited GVHD compared to historical controls (52). Indeed, these studies provide striking translational examples of the tolerogenic properties of human Tregs. Further applications of Tregs are anticipated as techniques for expansion are refined. Invariant Natural Killer T (iNKT) Cells: represent a very rare subset of T cells with restricted T cell receptors that additionally express NK cell markers. iNKT cells have now been implicated in diverse diseases including infection, autoimmunity, and tumor surveillance and appear to potentiate their immunosuppressive properties by expanding Tregs (53, 54). Low numbers of iNKT in the donor inoculum and delayed recovery after HCT have been inversely correlated with clinical GVHD (53, 55). More recent experiments in mice have shown infusion of donor iNKT cells, expanded in vitro, attenuated experimental GVHD by increasing Tregs in vivo (56). Thus, expanding iNKT (by in vivo or ex vivo manipulation) might provide another cellular mechanism to limit GVHD. Intracellular signaling pathways To direct immunologic responses that drive GVHD, complex extracellular signals must be transduced into networks that impact gene expression. Intracellular signaling pathways are again emerging as potentially novel targets for regulation of donor T cells

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and APCs. Although redundancy abounds, the JAK/STAT and NF-kB pathways are particularly promising in that they are understood to occupy central roles in mediating GVHD, in part by determining T cell function (Figure 3). Janus Kinase-2 (JAK2): is a non-receptor tyrosine kinase that associates with cell surface receptors to facilitate transmission of extracellular stimuli including cytokines (e.g. IL-6, IL 23) to signal transducer and activator of transcription (STAT) proteins which regulate transcription. Selective inhibition of JAK2 in vitro with the small molecule TG101348, administered during initial encounters between human T cells and myeloid DCs, initiated durable antigen specific tolerance (57). JAK2 inhibition correlates with increasing ratios of Tregs to effector T cells in vitro. Because JAK inhibitors are clinically approved for the treatment of myelofibrosis, they could be studied for the treatment and prevention of clinical GVHD. Indeed, a recent report indicated JAK1/2 blockage with ruxolitinib lessened GVHD related mortality in a murine model (58). In addition, JAK3 inhibition with tofacitinib (CP-690550) also attenuated GVHD in a MHC - disparate murine model, suggesting other JAKs may also be relevant (59). Spleen tyrosine kinase (Syk): is another non-receptor tyrosine kinase involved in signal transduction from TLRs. Syk inhibition with the clinically available fostamatinib reduced in vivo expansion of T cells from murine donors and mitigated experimental GVHD without affecting cytotoxic activity against leukemia targets or CMV (60). In APCs, fostamatinib reduced expression of co-stimulatory molecules after LPS and impaired their migration, while phagocytic activity remained intact.

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STAT3: Several inflammatory cytokines (IL-6, IL-21, and IL-23) converge in activating STAT3. In a chronic sclerodermatous model of GVHD, STAT3 deletion within CD4+ T cells limited pathologic evidence of sclerosis. Additionally, Tregs increased after STAT3 ablation, through thymus dependent and independent pathways (61). Further work has demonstrated that STAT3 is critical for T cell activation and acute GVHD (62, 63). nTregs become unstable following persistent STAT3 activation, but removal in these cells was insufficient to prevent GVHD. Surprisingly, deletion in naive CD4+ T cells was protective, suggesting that STAT3 may promote iTregs. Subsequent experiments using a small molecule inhibitor of STAT3 demonstrate increased phosphorylation of STAT5, demethylation of the key developmental transcription factor FOXP3 and iTreg expansion. Taken together, inflammatory cytokines regulate STATs which in turn determine T cell differentiation and function. However, the role of STAT signaling on modulating inflammatory gene programs may be divergent in different immune cell subsets, specifically in T cells and APCs. For example, STAT3 activation invokes tolerogenic effects upon APCs independent of phosphorylation (64). Treatment of murine bone marrow DCs with the histone deacetylase inhibitor (HDACi) vorinostat acetylated STAT3 and resulted in direct induction of indoleamine 2,3-dioxygenase (IDO) which prevented inflammatory responses. Nuclear factor NF-kB: signaling is a well-established mediator of the inflammatory response by regulating cytokine production, leukocyte recruitment, and cell survival (65). Despite the association of NF-kB with numerous inflammatory diseases, its role in GVHD has recently been appreciated. Transcription factors of alternative (RelB/p52) and canonical (c-Rel/p50) NF-kB pathways, normally sequestered in the cytoplasm of 14

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resting cells, accumulate in nuclei of APC and T cells upon activation. Experimentally, host DCs have increased expression of RelB in their nuclei after activation (66). Bone marrow chimeric mice deficient in RelB-/- have less early GVHD, while transfer of RelB-/donor bone marrow ameliorates late GVHD. Conventional DCs from RelB-/- animals are quantitatively reduced, induce less proliferation and produce less cytokines compared to wild-type counterparts. Similarly, activation of the alternate pathway can be accomplished by NIK (NF-kB inducing kinase) which promotes p52/RelB dimerization. Overexpression of NIK led to a rapid lymphocytic infiltration of multiple organs, while its deficiency prevented CD4+ T cell dependent GVHD (67). The effect of canonical NF-kB signaling by c-Rel, known to promote lymphocyte survival, was studied in MHC matched and mismatched models of GVHD. T cells lacking c-Rel induced minimal GVHD, but their GVT activity was preserved. These findings were later recapitulated in pre-clinical studies using a small molecule inhibitor of c-Rel (68). T Cell Bioenergetics: Targeting the altered bioenergetics of alloreactive T cells may be an alternate approach to treating GVHD. Although most cells employ aerobic glycolysis, the oxidative phosphorylation (OXPHOS) pathway is the primary generator of cellular ATP in proliferating lymphocytes (69). Murine data show that Bz-423, a benzodiazepine that inhibits mitochondrial F1F0-ATPase, led to apoptosis of alloreactive CD4+ and CD8+ donor T cells and improved GVHD related mortality. Fatty acids are an important fuel source during periods of OXPHOS (70). Selective inhibitors of OXPHOS induce apoptosis, particularly in CD4+ and CD8+ cells undergoing cell division in vitro.

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Regulation of Translation and Transcription. Translational regulation encompasses numerous overlapping yet divergent biologic processes that include posttranslational modifications (PTMs) such as phosphorylation, acetylation,

ubiquitination,

and

neddylation

that

regulate

immunity.

Likewise,

transcriptional regulation, particularly by epigenetics, are increasingly implicated in the immunobiology of GVHD. Inhibition of DNA methylation, histone acetylation and histone methylation can all induce tolerance. Because epigenetic states are modifiable, early phase trials with clinically available agents already are beginning to identify potential roles in GVHD prevention. Neddylation and the immunoproteosome: Although the ubiquitin-proteasome pathway is implicated in GVHD pathogenesis, the role of neddylation in regulating immune responses has recently been described. The neural precursor cell-expressed developmentally downregulated-8 (NEDD8) pathway operates within the ubiquitin-proteasome system by regulating several key PTMs. Inhibiting the transfer of NEDD8 to client proteins (termed neddylation) perturbs ubiquitination of proteins, many with oncogenic potential, and this pathway is currently being investigated in trials for AML with MLN4924, a first in class small molecule inhibitor of NEDD8 activating enzyme (NAE)(71). MLN4924 treatment of DCs markedly attenuated pro-inflammatory cytokine (TNFα and IL-6) production and T cell proliferation. Administration of MLN492 also directly inhibited T cell proliferation and GVHD in murine models (72). Mechanistic studies suggest regulation of immune responses by neddylation is associated with inhibition of NF-kB signaling. Of particular

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interest for clinical translation, MLN4924 exerted significantly greater in vitro suppression of DCs than corticosteroids or bortezomib, both with efficacy in GVHD. These findings may build on previous work showing proteasome inhibition with bortezomib prevented murine and human GVHD after HLA mismatched HCT (73, 74). Epigenetic modifiers of GVHD: DNA Methylation: Inhibition of DNA methyltransferase results in hypomethylation of promoters of several genes. Choi et al. demonstrated that in vitro and in vivo treatment with the hypomethylating agent azacitidine (Aza) caused inducible FOXP3 expression and other downstream epigenetic changes in CD4+CD25- T cells that impart suppressive function. This conversion of peripheral effector T cells in the presence of Aza mitigated GVHD while preserving GVL in models of MHC mismatched transplantation (75). Clinically, de Lima and colleagues demonstrated that Aza could be safely administered after HCT (76). Recently, Aza administered with alemtuzumab resulted in increased numbers of Tregs three months after HCT with intact cytotoxic T cell responses (77). Histone Acetylation: HDACi hyperacetylate histone proteins and have anti-neoplastic effects, but they possess potent immunosuppressive properties. Mechanistically, they regulate DCs and enhance Tregs. HDACi also reduced pro-inflammatory cytokines and co-stimulatory molecules that diminished experimental GVHD by increasing IDO expression (78). Furthermore, TLR agonists capable of inducing IDO reduced colonic injury (79). HDACi have variable effects on different HDACs. The key HDACs in regulating immunity remain unknown; therefore not all HDACi will likely have similar

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effects. This is exemplified by the observation that the potent HDACi (panobinostat and LBH589) accelerated GVHD in some models (80). Nonetheless, in a recent multi-center phase I / II trial, administration of vorinostat after reduced intensity conditioning mitigated GVHD in humans as in mice (81). We observed a day 180 grade II-IV GHVD incidence of 28%, however, several cases were limited to the upper GI tract GVHD resulting in a grade III-IV incidence of 12%. Although reduced intensity conditioning is associated with less anti-tumor effect, the incidence of relapse was not increased (16% at two years). Similar to pre-clinical observations, patients had significantly lower serum levels of IL-6 and TNF-α, but higher levels of IDO mRNA and absolute Tregs when compared to patients treated without vorinostat. Histone Methylation: adds yet another layer to the epigenetic landscape of GVHD. Enhancer of zeste homolog 2 (EZH2), a component of the polycomb repressive complex 2 (PRC2) functions to trimethylate histone H3 at lysine 27, which silences numerous genes including the pro-apoptotic protein Bim(82). Inhibition of EZH2 with the small molecule DZNep inhibited methylation of histones and prevented experimental GVHD; effects which were ameliorated in Bim knockouts. These observations point to epigenetic regulation as promoting unique immunomodulatory effects independent of Tregs or APCs. microRNA (miRs): While not classic epigenetic modifiers, miRs (non-coding RNA) are closely situated in that they direct translational repression and mRNA decay. Because microRNA-155 (miR-155) is up-regulated during T-cell activation, Ranganathan et al. examined this miRs role in murine GVHD, showing overexpression increased disease

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severity (83). Considering the extent of miRs in GVHD is unknown, but the role of APCs are well established, we undertook an unbiased analysis of miR expression in CD 11c+ DCs (84). Under LPS stimulation, miR-142 was highly expressed as was mRNA for IL6. Additional experiments revealed that miR-142 interacts with the 3’ untranslated region of IL-6 repressing its mRNA. Importantly, antagonism of miR-142 limits mortality from endotoxin induced sepsis (IL-6 was protective in this model). In complementary work, the laboratory is investigating the molecular mechanisms of miRs that attenuate T cell proliferation after allogeneic stimuli. Using a combined murine knockout and bioinformatics approach, two atypical E2Fs (transcription factors known to regulate cell cycling) were identified as targets of miR-142 (85). These data, together with the introduction of miR antagonists and mimetics into the clinic for diverse human diseases, suggest targeting miRs might provide hierarchical regulatory control over key pathways in GVHD. More recently, the AGO-CLIP method with subsequent ChIP microarray analysis was utilized to interrogate clinically relevant direct miR–mRNA interactions in T cells (86). Allogeneic T cells stimulated by DCs (compared to syngeneic or CD3/CD28 stimulated DCs) had a unique network of enriched miR–mRNAs, of which the previously unrecognized genes Wapal and Synj had the greatest differential expression. Wapal and Synj are regulated by multiple miRs and are specifically expressed following allostimulation in vitro. Their relevance to T cell responses was confirmed in vivo after Wapal and Synj specific knockdown attenuated GVHD severity in MHC disparate HCT. These experiments suggest the molecular programs of T cells likely differ in their

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response to alloreactive versus nominal antigenic stimuli. The specific functions of Wapal and Synj in T cells remain to be elucidated. CONCLUSION The prevention and treatment of GVHD without intensive immune suppression remains a major challenge for the HCT field. Over the past decade, we have witnessed a marked expansion in our understanding of the molecular underpinnings of GVHD. These advances provide novel opportunities to selectively mitigate GVHD while largely preserving vital immunologic function. The early clinical development of these approaches are summarized in Table 1. Not all observations merit translation since model systems cannot precisely mirror the genetic heterogeneity, concomitant immunosuppression and other complex variables inherent to clinical HCT. Still, insights from murine studies remain invaluable for understanding the biological principles that inform the next generation of acute GVHD studies. Indeed, a dynamic interchange among translational scientists and clinicians is necessary to optimally meld pre-clinical discoveries with well-designed trials that overcome this devastating complication.

ACKNOWLEDGEMENTS This work was supported by grants from the National Institutes of Health: AI-075284, HL-090775, and CA-173878 (P.R.); and ASBMT New Investigator Award (J.M.).

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Figure 1. Organizational schema for the diverse cellular and molecular mechanisms of acute GVHD.

Figure 2. Extracellular mediators and receptors implicated in promoting GVHD. Prototypical initiating events include chemotherapy and radiation associated tissue injury resulting in release of damage-associated and pathogen-associated molecular patterns (DAMPs / PAMPs) which regulate the activation APCs. In addition to lipopolysaccharide (LPS), several novel activating DAMPs / PAMPs such as heparin sulfate, uric acid, ATP, intestinal microbiota and their receptors have been described. These interactions, together with classic costimulatory signals (CD80 / CD86) modulate T cell receptor / MHC interactions and promote release of cytokines / chemokine (IL-6, IL-21, IL-23) that influence T cell differentiation, homing, and proliferation. MHC, major histocompatibility complex; TCR, T-cell receptor; CCR5, chemokine (C-C motif) receptor 5; P2X7R, purinergic receptor; nlrp3, multi-protein inflammasome; TLR, Toll-like Receptor; APC, Antigen presenting cell. Figure 3. Intracellular signaling and transcriptional / translational regulation. JAK / STAT and NF-kB are dominant pathways recently implicated in GVHD that amplify external stimuli directing transcription of genes involved in inflammation and regulation of T cell function. Upon translocating to the nucleus, transcription factors access target genes whose availability is controlled by the epigenetic status of the DNA and

27

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chromatin. Histone acetylation (Ac), histone methylation (M) and DNA methylation (Meth) have all been implicated in the pathogenesis of GVHD. Post translational modification with microRNAs, neddylation and ubiquitin-proteosome pathways also tightly regulate the resulting protein content of the cell, thus further influencing its propensity to divide and differentiate into an effector of GVHD. HAT, Histone acetyltransferases, HDAC, Histone deacetylases; DNMT, DNA methyltransferase; PRC2, Polycomb Repressive Complex 2.

28

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Table 1. Select targets for novel GVHD therapy with stage of development

Level of Action

Drug or Approach

Pre-clinical (animal model)

Clinical study (phase I-II)*

IL-6

Tocilizumab

+

+

IL-21

anti - IL-21 mAb

+

-

Maraviroc

+

+

Alpha-1-antitrypsin

+

+

cell adhesion

anti - DNAM-1

+

-

notch inhibition

anti DL-1, DL-4

+

-

tissue injury

P2X7 / Nlrp3 inhibition

+

-

microbiome

probiotic, other

+

+

Tregs / iNKT cells

+

+/-

JAK-2 / Syk inhibition

+

+/-

NF-kB

c-Rel inhibition

+

-

STATs

STAT3 inhibition

+

-

Bz-423

+

-

neddylation

MLN4924

+

-

microRNA

miR-142 antagonist

+

-

DNA methylation

Azacitidine

+

+

histone acetylation

Vorinostat

+

+

histone methylation

EZH2 inhibition

+

-

Extracellular cytokine / chemokine

CXCR5 other protease inhibition

cellular effectors Intracellular signaling pathways tyrosine kinase

T Cell bioenergetics post translational / epigenetic

*Based on published reports including absracts and listings at https://clinicaltrials.gov

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FIGURE 1.

EXTRACELLULAR

INTRACELLULAR

Mediators & Receptors

Signal Transduction

• • • • • •

Cytokines Chemokines DAMPs / PAMPs Cell Adhesion Microbiome Treg / iNKT

• • •

Transcription & Translation • • •

©

JAKs / STATs NF-κB Bioenergetics

Epigenetics MicroRNA PTMs


FIGURE 2. Tissue Damage (Chemotherapy / Radiaon) APC x

Cosmulatory molecule expression

x

Pro-inflammatory cytokine release

(+)

P2x7R

(+)

Nlrp3

(+)

(-)

GI-Tract

TLR

Siglecs

LEGEND DAMPs (e.g. ATP, Uric Acid, Heparin Sulfate)

CD80 /CD86 MHC

PAMPs (e.g. Bacteria / Microbiota)

Cytokines (e.g. TNFα, IL-6, IL-21, IL-23) Cytokine

TCR

CCR5

Receptors

T Lymphocyte x

↑ proliferaon

x

↑ Th1 / Th17 differenaon

x

↓ Treg expansion

x

↑ Migraon to GVHD target organs

©


FIGURE 3. IL6

TNF

IL6R

TNFR

JAK 2 P IkB P STAT 3

STAT 5

Neddylaon

NFkB

Ubiquinaon Proteasome

Cell Division / Differenaon

GVHD Target Genes

Epigenec Regulaon

Transcripon Meth

Ac

HAT / HDAC DNMT

Meth

PRC2

microRNA (miR-142)

M

T Lymphocyte

©


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