Enhancer of zeste homolog 2 (EZH2) is a part of the polycomb repressive complex and catalyzes the trimethylation of lysine 27 on histone H3 (H3K27me3). EZH2 inhibition has a complex role in the pathogenesis of acute myeloid leukemia (AML), in that it has been shown to be either a tumor suppressor or an oncogene depending on the stage of AML development and the genes that EZH2 is regulating during each stage. Unlike follicular and diffuse large B-cell lymphoma where EZH2 mutations result in gain of function, EZH2 mutations are typically loss of function in myeloid diseases. However, we hypothesized that in AML patients without EZH2 mutations, loss of EZH2 function may produce a phenotype that would allow for therapeutic targeting without influencing normal hematopoiesis. We used EPZ011989 (EPZ), an EZH2 inhibitor tool compound, to inhibit H3K27me3 in our studies. We started by treating the MOLM-13 AML cell line with EPZ and confirmed a decrease in H3K27me3. This reduction in H3K27me3 resulted in a slight decrease in metabolic activity via MTS assays as well as decreased colony formation in methocult. These studies were followed up with EPZ inhibition in primary AML samples in vitro. We found that EZH2 inhibition resulted in decreased self-renewal of primary AML samples but not of CD34+ bone marrow cells from normal donors. Furthermore, we found that after 7-day treatment with EPZ, primary AML samples undergo moderate differentiation as suggested by an increase in CD11b surface expression via flow cytometry. These results are further supported by the morphological changes seen after 14-days of EPZ treatment in vitro. Based on these results, we hypothesize that EZH2 inhibition in primary AML samples promotes the differentiation of AML blasts. Furthermore, our preliminary data suggests that daily treatment with 150 mg/kg of EPZ results in a survival advantage and reduced disease burden in the MOLM-13-luciferase murine xenograft model. Despite loss of function EZH2 mutations portending poor outcomes in myeloid malignancies, we demonstrate that pharmacologic EZH2 inhibition reduces AML blast stemness and promotes differentiation into mature myeloid cells. In contrast, no change in normal CD34+ stem cells occurs with EZH2 inhibition, offering the opportunity to selectively target myeloid leukemia. Citation Format: Sydney Fobare, Ola A. Elgamal, Emily H. Stahl, Abeera Mehmood, Jean Truxall, Mariah L. Johnson, Amina Abdul-Aziz, John C. Byrd, Erin Hertlein. EZH2 inhibition induces blast differentiation in acute myeloid leukemia [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 1824.
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.
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