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Acute graft-versus-host disease (GVHD) is the leading cause of non-relapse mortality following allogeneic hematopoietic cell transplantation. The majority of patients non-responsive to front line treatment with steroids have an estimated overall 2-year survival rate of only 10%. Bromodomain and extra-terminal domain (BET) proteins influence inflammatory gene transcription, and therefore represent a potential target to mitigate inflammation central to acute GVHD pathogenesis. Using potent and selective BET inhibitors Plexxikon-51107 and -2853 (PLX51107 and PLX2853), we show that BET inhibition significantly improves survival and reduces disease progression in murine models of acute GVHD without sacrificing the beneficial graft-versus-leukemia response. BET inhibition reduces T cell alloreactive proliferation, decreases inflammatory cytokine production, and impairs dendritic cell maturation both in vitro and in vivo. RNA sequencing studies in human T cells revealed that BET inhibition impacts inflammatory IL-17 and IL-12 gene expression signatures, and Chromatin Immunoprecipitation (ChIP)-sequencing revealed that BRD4 binds directly to the IL-23R gene locus. BET inhibition results in decreased IL-23R expression and function as demonstrated by decreased phosphorylation of STAT3 in response to IL-23 stimulation in human T cells in vitro as well as in mouse donor T cells in vivo. Furthermore, PLX2853 significantly reduced IL-23R+ and pathogenic CD4+ IFNγ+ IL-17+ double positive T cell infiltration in gastrointestinal tissues in an acute GVHD murine model. Our findings identify a role for BET proteins in regulating the IL-23R/STAT3/IL-17 pathway. Based on our preclinical data presented here, PLX51107 will enter clinical trial for refractory acute GVHD in a Phase 1 safety, biological efficacy trial.

Introduction T cell mediated acute graft-versus-host disease (aGVHD) is the main cause of non-relapse mortality in allogeneic hematopoietic cell transplant recipients. Protein arginine methyltransferase 5 (PRMT5) is a post-translational modifier and promotes activation of memory TH cells. We investigate mechanisms by which PRMT5 regulates T cell function and propose PRMT5 inhibition as a therapeutic strategy for aGVHD. Materials and Methods PRMT5 expression and function was evaluated in T cells of healthy and aGVHD mice and humans. We assessed T cell proliferation and effector function, using C220, a novel PRMT5 inhibitor. We tested effects of PRMT5 inhibition using in vivo mouse models of aGVHD, where mice received T cell depleted bone marrow + allogeneic splenocytes. In graft-versus-leukemia (GVL) experiments, P815 tumor cells were administered. Mice were treated with C220 or vehicle and monitored for survival and clinical aGVHD scores. Results PRMT5 expression and function is upregulated in T cells of mice and humans with aGVHD. Inhibition of PRMT5 reduces T cell proliferation, perturbs cell cycle and downregulates STAT-1 mediated interferon response. Administration of C220 improved survival in aGVHD mouse models and maintained tumor specific CTL response contributing to retention of Graft versus leukemia (GVL) effect. Conclusions Inhibition of PRMT5 using C220, down-regulates T cell proliferative and effector response, induces cell-cycle arrest, reduces interferon response and perturbs signaling pathways. C220 shows potent biological activity in vivo by reducing aGVHD clinical severity and prolonging survival in mouse models without compromising GVL. Therefore, PRMT5 is a novel and druggable target for aGVHD.

Introduction: The use of allogeneic hematopoietic cell transplantation to treat acute myeloid leukemia (AML) has risen in recent years. However, relapse remains the major cause of mortality post-transplant; while graft-versus-host disease (GVHD) - a T cell mediated immunological disorder is the major cause of non-relapse mortality. Dihydroorotate dehydrogenase (DHODH) supports T cell proliferation by playing a critical role in de novo pyrimidine synthesis and oxidative phosphorylation. We hypothesized that alloreactive T cells may rely on increased levels of pyrimidine pools and ATP to support rapid cell proliferation, making DHODH an interesting target to prevent GVHD. Additionally, DHODH inhibition is currently being pursued as a therapeutic option for AML. Therefore, we hypothesize that DHODH inhibition post-transplant will serve a dual purpose - i) target T cell metabolism to reduce GVHD and ii) prevent relapse due to direct anti-leukemic effects, thereby resulting in superior post-transplant outcomes. Methods: We tested the efficacy of a novel DHODH inhibitor (Cmpd 41), a lead clinical candidate, in preventing GVHD and retaining graft-versus-leukemia (GVL) effect. Human T cells isolated from PBMCs were activated with CD3/CD28 Dynabeads ± Cmpd 41. Cell proliferation (flow cytometry) and ATP production (Agilent Seahorse) was assessed. GVHD and GVL was assessedevaluated in a xenogeneic model where irradiated NSG mice received human PBMCS (~17x106 cells) and treated with vehicle or Cmpd 41 (10mg/kg, 2x/week) with addition of MOLM-13 cells (~1x104) for GVL. Splenocytes were harvested for analysis of cytokine production (intracellular flow cytometry). Results: DHODH inhibition with Cmpd 41 significantly reduced T cell proliferation and ATP production from both glycolysis and OXPHOS (fold change: Cmpd 41 vs. control- 0.56 and 0.68 respectively, p<0.01) compared to vehicle. DHODH inhibition significantly improved survival (mean survival: Cmpd 41 vs. vehicle: 56 days vs. 40 days, p<0.01) and reduced clinical scores (Cmpd 41 vs. vehicle: 1.8 vs. 4.2, p<0.05) compared to vehicle in the xenogeneic GVHD model. There was a significant reduction in IFN-γ and TNF-α cytokine producing T cells in the Cmpd 41 treated cohort compared to vehicle (Cmpd 41 vs. vehicle, IFN-γ: 8.33% vs. 31.28%, p<0.01; TNF-α: 1.37% vs. 27.25%, p<0.01). In a GVL model, mice that received both human PBMCs and Cmpd 41 showed decreased tumor growth and improved overall survival over mice given either treatment alone (p<0.01), showing that DHODH inhibition maintains GVL. Conclusion: DHODH inhibition is a novel approach to prevent and mitigate GVHD while retaining GVL effects. Combined with direct anti-leukemic effects, we propose that Cmpd 41 treatment in post-transplant relapse in the setting of past or active graft versus host disease will provide dual treatment of AML and GVHD thereby leading to improved patient outcomes. Citation Format: Kara M. Braunreiter, Lotus Neidemire-Colley, Natalie Sell, Yandi Gao, Sandip Vibhute, Chad Bennett, Ola A. Elgamal, Thomas Goodwin, Erin K. Hertlein, John C. Byrd, Parvathi Ranganathan. DHODH inhibition modulates T cell metabolism reducing GVHD and prevents relapse following allogeneic HCT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2335.