DHODH Inhibition Modulates T Cell Metabolism, Selectively Impairs Effector T Cell Response, Limiting Gvhd While Preserving GVL.

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Introduction Acute graft-versus-host disease (GVHD) is a donor T cell driven complication arising in patients receiving an allogeneic hematopoietic cell transplantation (allo-HCT). There is an urgent need for new strategies that limit GVHD while retaining the beneficial graft-versus-leukemia (GVL) effect. During GVHD , alloreactive T cells increase oxidative phosphorylation (OXPHOS) to meet the elevated metabolic demands required for proliferation and effector functions, including cytokine production . Dihydroorate dehydrogenase (DHODH), is essential for de novo pyrimidine biosynthesis and maintaining mitochondria membrane potential for OXPHOS, vital for T cell proliferation and effector function. Objective Evaluate DHODH inhibition as a strategy to prevent GVHD while retaining GVL effect . Methods For in vitro studies, CD3+ T cells were isolated via negative selection from healthy donor peripheral blood mononuclear cells (PBMCs) and activated with anti-CD3/CD28 dynabeads for 72 hrs. T cells were treated with HOSU-53 (10nM) either during activation (de novo), or post-activation for downstream analysis. Metabolic assays were performed on the Agilent Seahorse XFe96. We used a xenogeneic model of acute GVHD where PBMCs were injected into NSG mice , and recipient mice were treated with HOSU-53 (10mg/kg) or vehicle. We performed an in vivo GVL study using leukemic (MOLM-13) cells along with PBMCs injected into NSG mice . Results In vitro DHODH inhibition decreased T cell proliferation , reduced IFNγ (p<0.01), TNFα (p<0.05) cytokine production , and OXPHOS (p<0.005), without impacting viability, during de novo T cell activation compared to control. However, when T cells were treated with HOSU-53 post-activation, DHODH inhibition did not impact IFNγ or TNFα (p=0.5) cytokine production despite diminished OXPHOS (p<0.005), compared to control (Figure 1). In vivo, HOSU-53 treatment reduced disease severity and improved survival in a xenogeneic model of acute GVHD (p<0.01). Enumeration of T cells infiltrating the liver showed significantly diminished pathogenic TBET+,IFNγ+ Th1 cells , Rorγt+, IL-17+ Th17, TNFα+ CD4 T cells as well as IFNγ+ CD8 T cells (p<0.05) in HOSU-53 treated mice compared to vehicle. Interestingly, absolute counts of CD25+, Foxp3+ Tregs were decreased, accompanied by an increase in double negative (DN, CD4-,CD8-) T cells in HOSU53 treated mice compared to vehicle (p<0.05, Figure 2). Importantly, DHODH inhibition preserves the beneficial GVL effect, improving survival (p<0.0001) in a xenogeneic GVL model (Figure 3). Conclusions DHODH inhibition selectively diminishes naive T cell activation, expansion and effector function while expanding the DN population. Thus, DHODH inhibition is a novel strategy to limit GVHD that may provide added benefit of promoting DN T cell mediated immune suppression , which has been correlated with reduced risk of aGVHD.

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