DHODH inhibition alters T cell metabolism limiting acute graft-versus-host disease while retaining graft-versus-leukemia response

Abstract

Acute graft-versus-host disease (GVHD) is a donor T cell driven complication and the leading cause of non-relapse mortality in patients receiving an allogeneic hematopoietic cell transplantation (allo-HCT). Allogeneic donor T cells eradicate residual leukemia and prevent relapse via the graft-versus-leukemia (GVL) effect and are critical for responding against opportunistic infections post-transplant. Current regimens successful in preventing GVHD are broadly immunosuppressive and come at the cost of increased risk of relapse and/or infection. Therefore, there is an urgent need for new approaches that limit GVHD while retaining GVL responses.

During GVHD, alloreactive T cells boost their energy production through oxidative phosphorylation (OXPHOS) and glycolysis, supporting heightened proliferation and pathogenicity against healthy host tissues. The enzyme dihydroorate dehydrogenase (DHODH), is essential for de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential during OXPHOS. Having shown upregulation of DHODH messenger RNA and protein expression in activated human T cells, we evaluated DHODH inhibition, via a small molecule inhibitor HOSU-53, as a therapeutic approach for GVHD. Inhibiting DHODH significantly reduced oxidative metabolism in T cells both during and after activation, while selectively suppressing inflammatory cytokine production in de novo activated, but not previously activated, T cells. In a xenogeneic model, HOSU-53 treatment limited GVHD severity, decreased pathogenic Th1 and Th17 response, and preserved beneficial GVL effects. Altogether, we identify DHODH inhibition as an innovative treatment strategy in allo-HCT recipients to reduce GVHD severity and retain effective GVL response.

Introduction

Over 8,000 allogeneic hematopoietic cell transplants (allo-HCT) are performed annually in the U.S. and represent the only curative therapy for many high-risk hematological malignancies.^1–5 In patients with acute myeloid leukemia (AML), myelodysplastic syndromes, and acute lymphoblastic leukemia, an allo-HCT replaces the dysfunctional, malignant hematopoietic system with stem cells from an HLA-matched healthy donor while donor immune cells eliminate residual disease via the beneficial graft-versus-leukemia (GVL) effect.^6–8 Allogeneic T cells can also recognize the recipient tissues as foreign through major and/or minor HLA mismatching, causing a severe immunological complication known as acute graft-versus-host disease (GVHD).^6,7,9–12 Despite advances in donor matching, conditioning regimens, and prophylactic strategies, between 30% and 70% of allo-HCT patients develop acute GVHD which remains a leading cause of non-relapse mortality.^13,14 The recipients’ conditioning regimen causes cellular apoptosis and breach of the intestinal barrier releasing pathogen- and damage- associated molecular patterns as well inflammatory chemokines and cytokines.^9,15 In this inflammatory milieu, recognition of recipient allo-antigens by donor T cells results in activation, expansion, and migration of alloreactive donor T cells to tissues such as the skin, liver, and GI tract, with severe cases resulting in end-organ damage and possible death of the recipient.^9,15

Prophylactic regimens for GVHD commonly consist of combinations of calcineurin inhibitors and antimetabolites.^16,17 Recent clinical trials have shown that using post-transplant cyclophosphamide (PTCy) as prophylaxis reduces the occurrence of both acute and chronic GVHD, without a significant impact on the relapse rate.^16,18–20 Extensive preclinical^21–23 and clinical²⁴ investigations into the role of CD28 co-stimulation in T cell alloreactivity have resulted in FDA approval for abatacept, a selective T cell costimulatory modulator. Abatacept has been effective in reducing the incidence of severe Grade III to IV acute GVHD, although it does not substantially improve the rates of chronic GVHD or the occurrence of relapse and viral infections.^25–28 Despite continuous improvements in GVHD prophylaxis, approximately 52% of allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients still develop Grade II to IV GVHD, subsequent treatments remain suboptimal, and 15% of patients experience severe GVHD with poor outcomes.^9,29–34

During GVHD, alloreactive T cells upregulate both oxidative phosphorylation (OXPHOS) and glycolysis leading to increased proliferation and cytolytic activity against healthy host tissues.^35–39 Targeting these metabolic processes post allogeneic transplant has shown promise in alleviating acute GVHD while preserving beneficial GVL effects in mouse models.^36,40,41 In vivo, T cells deficient in mitochondrial electron transport chain (ETC) complexes III or IV fail to proliferate after antigen exposure.^42–45 Interestingly, impairing ETC function does not interfere with thymic development^42,43 as these T cells meet their energy demands through glycolysis and metabolite generation through other redundant mechanisms. Therefore, targeting mitochondrial membrane potential and ETC function could be a viable strategy to mitigate GVHD without impairing GVL function of the donor T cells.

The rate-limiting enzyme dihydroorate dehydrogenase (DHODH) is involved in de novo pyrimidine biosynthesis and in maintaining mitochondrial membrane potential for the ETC, processes vital for T cell proliferation and effector function. DHODH donates an electron to ubiquinone causing its’ transfer to ubiquinol. ETC complex III oxidizes ubiquinol back to ubiquinone, maintaining function of complexes I and II.⁴⁶ Inhibition of DHODH has been identified as a promising therapeutic strategy for T cell mediated autoimmune disorders such as multiple sclerosis (MS)^47,48 and amyotrophic lateral sclerosis⁴⁹ as well as for treating hematological malignancies such as Acute Myeloid Leukemia.⁵⁰ In MS, DHODH promotes expansion of T cells bearing high-affinity TCRs, and inhibiting DHODH function, vis-a-vis teriflunomide, corrects metabolic disturbances in T cells, subsequently alleviating disease.⁵¹ HOSU-53 is a potent DHODH inhibitor, with nanomolar inhibitory capacity and pharmaceutical properties enabling once daily oral dosing. Most importantly, HOSU-53 has demonstrated therapeutic efficiency in murine models of AML.⁵² However, the relevance of DHODH function and therapeutic potential within the context of GVHD is largely unknown. In this manuscript, we evaluated DHODH inhibition, using the pharmacological inhibitor (HOSU-53), as a novel strategy to mitigate both acute GVHD and relapse in xenogeneic models of GVHD and leukemia.

Materials and methods

Mice

NSG mice were purchased from the OSUCCC Target Validation Shared Resource (Columbus, Ohio, USA). For transplant experiments, recipient mice were between 12 and 16 wks of age. All animal studies were conducted in accordance with the rules and regulations of the Institutional Animal Care and Use Committee at OSU.

Agilent seahorse ATP rate assay

Metabolic assays were performed on the Agilent Seahorse XFe96 following manufacturer’s instructions and using all recommended reagents supplied by the manufacturer. Briefly, plates were coated with Cell-Tak at 22.4 µg/ml (Corning 354240) and neutralized with 0.1M sodium bicarbonate, incubated for 20 minutes at room temperature, then washed twice with sterile water. T cells were suspended at 40x10⁵ cells/mL in XF Assay Media [Seahorse XF DMEM Media, pH 7.4 (Agilent 103575-100), 10 mM Seahorse XF Glucose (1.0 M stock, Agilent 103577-100), 1 mM Seahorse XF Pyruvate (100 mM stock, Agilent 103578-100), 2 mM Seahorse XF L-Glutamine (100 mM stock, Agilent 103579-100)] and plated at 200,000 cells/well in the coating 96-well plate. Cells were adhered to the plate by centrifugation prior to the addition of more XF Assay Media to bring wells to a total volume of 180 µl. For the Mito Stress Test (Agilent 103015-100), Oligomycin, FCCP, and Rotenone + Antimycin A were suspended in XF Assay Media to a concentration of 15 µM, 5 µM, and 5 µM, respectively, and loaded into the hydrated sensor cartridge at volumes of 20 µl, 22 µl, and 25 µl, respectively. For the Glyco Stress Test (Agilent 103020-100), Glucose, Oligomycin, and 2-DG were suspended in XF Assay Media to a concentration of 10 µM, 1 µM, and 50 µM, respectively, and loaded into the hydrated sensor cartridge at volumes of 20 µL , 22 µL, and 25 µL, respectively. Assays was performed using the Wave Software (Agilent) and data analyzed using Seahorse Analytics (Agilent).

qRT-PCR

RNA was isolated from 0.5-2x10⁶ cells using Trizol (Invitrogen 15596026) and Phasemaker Tubes (Invitrogen A33248) and cDNA was made from 500 ng of RNA using the RevertAid RT Reverse Transcription Kit (Thermo Scientific K1691), following manufacturer’s instructions. qRT-PCR was performed using TaqMan Real-Time PCR Assays (Applied Biosystems) and TaqMan Universal PCR Master Mix (Applied Biosystems 4304437) with 1 µl cDNA (5 µl of a 1:5 dilution loaded per well). Individual assays used are: DHODH (Hs00361406_m1), Hprt1 (Hs02800695_m1), SDHA (Hs00188166_m1), and UBE2D2 (Hs00366152_m1).

Western blot

Cells were lysed in RIPA buffer (Pierce) with protease inhibitor cocktail (Millipore: 539131-1VL) Protein was quantified using the Pierce Rapid Gold BCA Protein Assay Kit (Thermo Scientific A53225). 20 µg of total protein was loaded per well and transferred to nitrocellulose membrane. Primary antibodies for DHODH (CST no. 26381) and Vinculin (CST no. 13901) were incubated at 4 °C overnight and secondary antibodies were incubated for 1 hour at room temperature (IRDye 680RD goat anti-rabbit (Li-Cor 926-68071) and IRDye 800CW donkey anti-mouse (Li-Cor 926-32212), both 1:20,000). Fluorescence was detected using the Li-Cor Odyssey CLx and band intensity quantification was performed using the ImageStudio software (Li-Cor).

Cell culture and flow cytometry

Human T cells were isolated from buffy coats (Versiti Indiana) using the Pan T cell Isolation Kit (Miltenyi Biotec). Cells were stimulated with CD3/CD28 Dynabeads (Invitrogen) for 48 to 72 h ± HOSU-53, followed by incubation for 5 hours with Cell Stimulation Cocktail (eBioscience) and Protein Transport Inhibitor (Thermo Fisher Scientific). Cells were then stained with LIVE/DEAD fixable cell viability dye (Thermo Fisher Scientific) and surface antibodies, permeabilized and fixed, followed by staining with intracellular antibodies and analyzed within 24 h. For post activation studies, human T cells were stimulated first with CD3/CD28 Dynabeads for 72 h. Beads were subsequently removed, and cells rested and expanded with rhIL-2 (Peprotech) ± HOSU-53 for 72 h. On the day before metabolic/flow cytometry analysis, cells were restimulated with CD3/CD28 Dynabeads overnight. Data was acquired using a spectral Cytek Aurora flow cytometer, and analysis performed using FlowJo (Tree Star).

Xenogeneic GVHD model

Mice were transplanted under standard protocols approved by the University Committee on Use and Care of Laboratory Animals at OSU. Only age- and sex-matched mice were used for transplant experiments. Briefly, NSG mice were irradiated with 100 cGy one day before transplant. Fresh human PBMCs (1.7-2.0 x10⁷) were administered on the day of transplant by tail-vein injection. Recipients were treated with vehicle or DHODH inhibitor HOSU-53 (10 mg/kg), administered by oral gavage twice weekly starting at day +1 post-transplant until the end of the study.

Clinical assessment of acute GVHD

Recipient mice were weighed 2 to 4 times per week and monitored daily for clinical signs of acute GVHD and survival. A scoring method adapted and modified from Cooke et al.⁵³ was used to assess clinical changes associated with acute GVHD. Briefly, this scoring system incorporates 5 clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity. Individual mice were ear tagged and graded (on a scale from 0 to 10) 3 times a week. Mice who reached an acute GVHD score of more than or equal to 7 were euthanized and their tissues harvested. A separate cohort of mice were euthanized on day 40 post-transplant and single cell suspensions from the spleen and liver were generated via manual homogenization and liver dissociation kit, (Miltenyi no. 130105807) respectively, and assessed by flow cytometry. The histological intensity of acute GVHD in different liver sections was assessed on a 28-point scale by evaluating 7 parameters comprising portal tract inflammation, bile duct injury, vascular endotheliitis, periportal, or limiting plate necrosis, lobular necro-inflammatory activity, zonal necrosis, and sinusoidal lymphocytosis.⁵⁴

Xenogeneic GVL model

NSG recipients were irradiated (100 cGy) on day −1. Firefly luciferase and GFP transduced MOLM-13 acute myeloid leukemia cells (0.5 × 10⁶) were injected intravenously on day 0. Human PBMCs (1.7–2.0 ×10⁷) were injected intravenously on day +1. Treatment groups included vehicle and HOSU-53 10 mg/kg, administered by oral gavage twice weekly starting day +2 post-transplant. Molm-13 and human PBMCs alone served as the control groups. Molm-13-induced death was defined by hind-limb paralysis. GVHD death was defined as the absence of leukemia and presence of clinical and histopathological signs of GVHD.

In vivo imaging

Xenogen IVIS imaging system (Caliper Life Sciences) was used for live animal imaging. Mice were anesthetized using 1.5% isofuorane (Piramal Healthcare). XenoLight RediJect D-Luciferin Ultra Bioluminescent Subtrate (150 mg/kg body weight; 30 mg/ml in PBS; Perkin Elmer) was injected intraperitoneally and IVIS imaging was performed 7 to 10 min after substrate injection. Whole body bioluminescent signal intensity was determined using IVIS Living Image software v4.3.1 (Caliper Life Sciences), and pseudo-color images overlaid on conventional photographs are shown. Data were analyzed and presented as photon counts per area.

Degranulation assay

CD8 T cell degranulation was measured by intracellular production of IFN-γ and CD107a in response to ex vivo PMA/ionomycin stimulation. Splenocytes harvested from vehicle and HOSU-53 treated mice were stimulated and stained for intracellular cytokines as described above. Donor T cells were stained with human CD45 for identification. Cells were analyzed by flow cytometry.

Statistical analysis

Survival data were analyzed using Kaplan-Meier curves and statistics analyzed via log-rank test. Student unpaired t-test was used for all statistical analysis except where indicated. One-way ANOVA with Dunnet post-hoc test was used for comparisons with greater than 2 groups. For estimating statistical significance in clinical scores using multiple t-tests over time, P-values were adjusted using the 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli.⁵⁵ Data represent mean ± SD. All analyses were performed using GraphPad Prism 9.0. *P < 0.05, **P < 0.01, ***P < 0.001, NS non-significant.

Results

DHODH inhibition reduces T cell proliferation and function during de novo activation

Activated human T cells upregulate DHODH as evidenced by a significant increase in both DHODH mRNA (Fig. 1A) and protein expression (Fig. 1B, C) following CD3/CD28 stimulation. To investigate the impact of DHODH inhibition on T cell proliferation and viability, cell-trace violet labeled T cells were stimulated with CD3/CD28 beads in the presence of a range of HOSU-53 (0–1,000 nM). We observed a 50% reduction in T cell proliferation at 10 nM and 100 nM (Fig. 1D, E); however, we also observed reduction in T cell viability at 100 nM (Fig. 1F). Thereby, we selected 10 nM as the optimal concentration for further in vitro studies.

In corroboration with the proliferation studies, we observed a decrease in Ki67 expression across both CD4+ and CD8+ compartments of T cells following HOSU-53 treatment (Fig. 2A). Furthermore, DHODH inhibition also significantly reduced IFN-γ and TNF-α production, indicating decreased inflammatory function (Fig. 2B, C). Given DHODH localization in the mitochondria, and its’ contribution to mitochondrial membrane potential,⁵⁰ we assessed OXPHOS and glycolysis in T cells treated with HOSU-53. Inhibition of DHODH decreased mitochondrial respiration, diminishing both maximal respiration and spare respiratory capacity (Fig. 2D, E), while also significantly reducing glycolysis and glycolytic capacity (Fig. 2F, G). To test whether the observed metabolic impairment was independent of the role of DHODH in de novo pyrimidine synthesis, we conducted a uridine rescue assay. Our results show that HOSU-53 mediated reductions in OXPHOS and glycolysis were rescued in the presence of uridine supplementation (Fig. S1).

Our studies so far investigated the impact of DHODH inhibition when used concurrently with T cell activation. To investigate whether DHODH inhibition could modulate the function of previously activated T cells, we first stimulated T cells and then treated with HOSU-53. Interestingly, in this setting we found that DHODH inhibition reduced only OXPHOS (Fig. 3A, B), with no impact on glycolysis and glycolytic capacity (Fig. 3C, D). Further, there was no difference in Ki67 expression, IFN-γ, or TNF-α cytokine production in previously activated T cells (Fig. 3E–G). This suggests that DHODH inhibition preferentially targets T cell function during de novo activation without impacting the phenotype of already activated T cells.

DHODH inhibition reduces in vivo GVHD severity

To evaluate the therapeutic potential of DHODH inhibition, we utilized a xenogenic model of acute GVHD wherein NSG mice were transplanted with human PBMCs and treated with HOSU-53 or vehicle control. DHODH inhibition via HOSU-53 significantly extended survival (Fig. 4A, median survival 56 vs. 40 days, HOSU-53 vs. control, P < 0.01) and decreased GVHD severity, as observed by reduced clinical scores (Fig. 4B). We found that although mice under DHODH inhibition eventually succumbed to GVHD, HOSU-53 treatment significantly reduced liver damage compared to vehicle treatment alone (Fig. 4C, D). Furthermore, HOSU-53 also reduced pro-inflammatory IFN-γ and TNF-α cytokine production by donor CD4+ T cells taken from the spleen (Fig. S2A, B). To further investigate this pathology, we conducted a separate experiment using the same xenogeneic GVHD model, but created single cell suspensions of whole liver tissue at day 40 post allo-HCT for FACS analysis. In this case, we found decreased absolute numbers of inflammatory Th1, Th17, and TNF-α+ CD4 T cells infiltrating the liver (Fig. 4E–G) as well as a decrease in Treg numbers (Fig. 4H). To investigate whether DHODH inhibition impacted antigen presentation, we evaluated MHCII expression on donor and recipient splenic antigen presenting cells and found no differences between treatment cohorts (Fig. S3A–C). Altogether, these data indicate that DHODH inhibition in human T cells provides protection from acute GVHD disease progression and lethality in vivo.

HOSU-53 does not impair the beneficial GVL effect in allo-HCT

Relapse prevention serves as the main purpose of allogenic transplants in leukemia-bearing patients. We therefore investigated whether HOSU-53 preserved anti-leukemic effect of donor T cells against AML. In vitro, HOSU-53 treatment retained anti-tumor T cell efficacy (Fig. S4B). To investigate this effect in vivo, we used a xenogeneic model of GVL wherein NSG mice were infused with a GFP+ luciferase transduced FLT3-ITD+ MOLM-13 AML cell line along with PBMCs and subsequently treated with HOSU-53 or vehicle. We observed an overall improvement in median survival in mice receiving both PBMCs and HOSU-53 treatment (Fig. 5A, B, median survival 29 vs. 24 d, HOSU-53 vs. control, P < 0.0001). To help assess for cause of death, tumor burden was measured via flow cytometric evaluation of GFP+ MOLM-13 cells in the spleen at end of study. This analysis showed comparable levels of leukemic cells in the spleen in mice that received either MOLM-13 alone or MOLM-13 plus HOSU-53 (∼70% GFP+ MOLM-13 cells). In contrast, mice receiving PBMCs, with or without HOSU-53 treatment, eradicated GFP+ MOLM-13 tumor cells equally, showing retention of beneficial GVL effects in the presence of HOSU-53 (Fig. 5B, C). DHODH inhibition also retained IFN-γ and TNF-α production by CD8+ and CD4+ T cells, likely contributing to their anti-tumor function (Fig. 5D–G).

Discussion

Agents that prevent GVHD without increasing the risk of relapse remain elusive in the post-allo-HCT landscape. Using HOSU-53, a novel small-molecule inhibitor with favorable oral bioavailability, we show that DHODH inhibition prevents GVHD while retaining the GVL effects in human T cells. Our results help to better understand the therapeutic effects of pyrimidine synthesis inhibitors post allo-HCT and we anticipate that the knowledge gained in this preclinical work will provide further support for the clinical translation of DHODH inhibitors that selectively influence effector T cell function.

Nucleotide synthesis inhibitors such as mycophenolate mofetil and methotrexate are commonly used as GVHD prophylaxis strategies.⁵⁶ Despite this treatment, infections and GVHD are two of the most common reasons for hospitalization of allo-HCT recipients,⁵⁵ possibly due to the broad immunosuppressive action of these agents with nonspecific reductions in both effector and memory T cells.⁵⁷ We initially became interested in studying the DHODH pathway due to selective retention of infectious immune response in MS patients who received teriflunomide, a DHODH inhibitor.⁵⁸ Inhibiting DHODH in MS mouse models showed decreased clonal expansion and effector function of high-affinity T cells.^47,48 Thus, we posited that inhibition of DHODH, and its’ differential impact on T cell subsets,^59–61 might be an effective strategy to mitigate GVHD while retaining GVL responses.

Our results demonstrate that DHODH expression is upregulated in activated T cells and that disruption of DHODH modulates multiple components of T cell effector function, including proliferation and inflammatory TNF-α and IFN-γ cytokine production during de novo activation. Furthermore, DHODH inhibition results in diminished T cell metabolic function, with decreased mitochondrial respiration and glycolysis. Interestingly, T cells that receive DHODH inhibition post-activation still retain proliferative capability, as evidenced by Ki67 expression, as well as inflammatory TNF-α and IFN-γ production, despite a major lesion in OXPHOS. These results suggest that DHODH is selectively integral to the development of robust effector T cells, consistent with results in the literature where DHODH inhibition selectively blocked T differentiation into an effector phenotype.⁶¹ Interestingly, we observed a rescue of OXPHOS and glycolytic function in T cells treated with HOSU-53 and supplemented with uridine, suggesting that the impact of DHODH inhibition on oxidative metabolism is downstream of nucleotide starvation, in corroboration with previously published research.⁵¹

While it is known alloreactive T cells rely heavily on OXPHOS and aerobic glycolysis during early activation, fully understanding the metabolic processes within GVHD remains a work in progress. Previous work has shown that early glucose uptake by alloreactive T cells leads to enhanced effector function and tissue damage. These T cells rely on increased mitochondrial respiration and are vulnerable to ETC manipulation.³⁵ Molecular inhibition of glycolysis selectively suppresses alloreactive T cells, and genetic ablation of glucose uptake in donor T cells prevents GVHD while retaining GVL.⁴⁰ Furthermore, Inhibiting ATPase in mitochondrially active T cells improves survival, lowers clinical scores, and decreases lymphocytic infiltration into target organs in GVHD.⁴¹ Notably, under DHODH inhibition, we observed decreased Th1 (CD4+TBET+IFN-γ+), Th17 (CD4+Rorγt+IL17+) and CD4+TNF-α T cells within our GVHD model. Furthermore, Treg (Foxp3+, CD25hi) cells, well known to rely heavily on OXPHOS for their metabolic needs,^62–64 were similarly diminished following HOSU-53 treatment, suggesting that increased tolerance was not likely to be the mechanism underlying the improved survival benefit. Interestingly, we observed increased CD8+IFN-γ+ T cells within our GVL model. Congruently, others have commented on the differential metabolic profiles between CD4+ and CD8+ T cells, and metabolic manipulation leading to diminished GVHD pathogenesis without significantly impacting GVL.⁶⁵ However, these previous in-vivo research has been limited to murine models, without much investigation on impact on human T cells. Here, using a human PBMC into NSG model of xenogeneic GVHD, we identify DHODH as a contributor to effector function in human T cells during acute GVHD pathogenesis. Furthermore, this metabolic manipulation retains the necessary GVL responses post allo-HCT. HOSU-53 treatment alone is insufficient at clearing tumor at the administered dose of 10 mg/kg twice weekly, but it also does not disrupt the ability of PBMCs to clear allogeneic tumor cells. To note, this 10 mg/kg twice weekly is a much lower dose than what has been previously reported as required for maximal survival in an AML model (10 mg/kg daily or 30 mg/kg twice weekly).⁵² This infers that the survival benefit seen with concurrent PBMC and HOSU-53 administration is likely attributable to a retained GVL effect versus any direct anti-leukemic effect of DHODH inhibition.

Recently, allo-HCT phase II clinical trials evaluating efficacy of naïve T cell depleted allografts revealed very low incidence of acute GVHD along with no chronic GVHD and no apparent risk of relapse or non-relapse mortality.⁶⁶ Supporting these data, murine models of allo-HCT showed that in the absence of naïve T cells, CD4+CD25- memory T cells do not cause GVHD⁵⁹ while CD8+ memory and naïve T cells have equivalent GVL capabilities.⁶⁰ Concurrently, DHODH was found to be integral to the development of robust effector T cells. A caveat of the xenogeneic GVHD model is that it is not optimal for evaluating memory responses, however, previously published research has shown that inhibition of DHODH in patients with MS selectively blocked CD8+ T cell differentiation into an effector phenotype while retaining memory T cell responses against infectious agents.⁶¹ Therefore, pharmacologically inhibiting DHODH could selectively inhibit naïve alloreactive T cell activation and induce differentiation into memory T cells thereby limiting GVHD severity while preserving GVL.

In conclusion, HOSU-53 is a potent DHODH inhibitor, impacting T cell metabolism and immune function. DHODH inhibition post-transplant increases recipient survival and decreases GVHD severity, without impairing human T cell anti-leukemic function in a xenogeneic model. Our results, along with the reported direct anti-leukemic effects of HOSU-53,⁵¹ provide a strong rationale for the use of DHODH inhibition post allo-HCT to prevent acute GVHD while minimizing risk of relapse.

Acknowledgments

This research was made possible through resources, expertise, and support provided by the Pelotonia Institute for Immuno-Oncology (PIIO), which is funded by the Pelotonia community and the OSUCCC. We thank the PIIO and the Immune Monitoring and Discovery Platform for spectral flow cytometry and Seahorse assays. We thank the Target Validation Shared Resource (TVSR) and the Drug Discovery Shared Resource (DDSR) at the Ohio State University Comprehensive Cancer Center for providing the NSG mice and HOSU-53, respectively, used in the preclinical studies described herein. TVSR and MCSR are NCI supported shared resources through the OSUCCC grant P30 CA016058. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Health.

Author contributions

R.K. performed T cell isolations, seahorse assays, western blots, in vitro and ex vivo FACS analysis, in vivo GVHD models, analyzed the data and wrote the manuscript. K.M.B. and L.N.C. performed the T cell isolations, seahorse assays, ex-vivo FACS analysis, real-time PCR, and in vivo GVHD and GVL models. N.S and Y.G. performed T cell isolations, in-vitro T cell assays and in-vivo imaging. C.B. and S.V. provided the HOSU-53 drug. C.B., C.A.B., O.A.E, T.G, E.H, H.K.C., and J.C.B. provided ideas, discussion, and edited the manuscript. P.R designed the study, supervised research, analyzed and interpreted the data, and edited the manuscript.

Supplementary material

Supplementary material is available at The Journal of Immunology online.

Funding

This work was supported by The OSUCCC Drug Development Institute, the Paula and Rodger Riney Foundation, OSU CCC and Division of Hematology start-up funds, NIH R01CA252469, R01HL163849, American Cancer Society Research Scholar Grant RSG-22-053-01-IBCD (P.R.); NIH T32CA090223 fellowship to K.B. Research reported in this publication was supported by the Ohio State University Comprehensive Cancer Center and the National Institutes of Health under grant number P30 CA016058.

Conflicts of interest

J.C.B., T.E.G., O.A.E., E.K.H., C.B., and S.V. are inventors on the patent for HOSU-53. H.K.C. has served on advisory boards or consulting for Incyte, Sanofi, and Actinium and research support from Opna. The remaining authors declare no competing financial interests.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

References

Author notes