Eltrombopag inhibits TET dioxygenase to contribute to hematopoietic stem cell expansion in aplastic anemia
Yihong Guan, Metis Hasipek, Dongxu Jiang, Anand D. Tiwari, Dale R. Grabowski, Simona Pagliuca, Sunisa Kongkiatkamon, Bhumika Patel, Salendra Singh, Yvonne Parker, Thomas LaFramboise, Daniel Lindner, Mikkael A. Sekeres, Omar Y. Mian, Yogen Saunthararajah, Jaroslaw P. Maciejewski, Babal K. Jha
Yihong Guan, Metis Hasipek, Dongxu Jiang, Anand D. Tiwari, Dale R. Grabowski, Simona Pagliuca, Sunisa Kongkiatkamon, Bhumika Patel, Salendra Singh, Yvonne Parker, Thomas LaFramboise, Daniel Lindner, Mikkael A. Sekeres, Omar Y. Mian, Yogen Saunthararajah, Jaroslaw P. Maciejewski, Babal K. Jha
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Research Article Hematology

Eltrombopag inhibits TET dioxygenase to contribute to hematopoietic stem cell expansion in aplastic anemia

Abstract

Eltrombopag, an FDA-approved non-peptidyl thrombopoietin receptor agonist, is clinically used for the treatment of aplastic anemia, a disease characterized by hematopoietic stem cell failure and pancytopenia, to improve platelet counts and stem cell function. Eltrombopag treatment results in a durable trilineage hematopoietic expansion in patients. Some of the eltrombopag hematopoietic activity has been attributed to its off-target effects, including iron chelation properties. However, the mechanism of action for its full spectrum of clinical effects is still poorly understood. Here, we report that eltrombopag bound to the TET2 catalytic domain and inhibited its dioxygenase activity, which was independent of its role as an iron chelator. The DNA demethylating enzyme TET2, essential for hematopoietic stem cell differentiation and lineage commitment, is frequently mutated in myeloid malignancies. Eltrombopag treatment expanded TET2-proficient normal hematopoietic stem and progenitor cells, in part because of its ability to mimic loss of TET2 with simultaneous thrombopoietin receptor activation. On the contrary, TET inhibition in TET2 mutant malignant myeloid cells prevented neoplastic clonal evolution in vitro and in vivo. This mechanism of action may offer a restorative therapeutic index and provide a scientific rationale to treat selected patients with TET2 mutant–associated or TET deficiency–associated myeloid malignancies.

Authors

Yihong Guan, Metis Hasipek, Dongxu Jiang, Anand D. Tiwari, Dale R. Grabowski, Simona Pagliuca, Sunisa Kongkiatkamon, Bhumika Patel, Salendra Singh, Yvonne Parker, Thomas LaFramboise, Daniel Lindner, Mikkael A. Sekeres, Omar Y. Mian, Yogen Saunthararajah, Jaroslaw P. Maciejewski, Babal K. Jha

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Figure 3

Epag treatment mimics loss of Tet2, and expansion of myeloid compartment is Tet2 dependent.

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Epag treatment mimics loss of Tet2, and expansion of myeloid compartment...
(A) Epag dose response in the CFUs of Tet2+/+ murine bone marrow cells. (B) Epag increased CFUs in Tet2+/+ but not Tet2+/– and Tet2–/– cells. Data of the second plating are shown. (A and B) Six mice per group/treatment were used in 2 independent experiments. (C and D) Epag significantly increased neutrophil and monocyte count in vivo in Tet2+/+ but not in Tet2–/– graft recipient mice. Tet2+/+ CD45.1, Pep Boy mice were lethally irradiated prior to the transplant of 2 million Tet2+/+ or Tet2–/– bone marrow cells (CD45.2) via tail vein injection. Peripheral blood samples were counted by Hemavet. (E) Gating strategy of flow cytometry analysis for HSPCs, Lin–Sca-1+c-Kit+ (LSK), Lin–Sca-1–c-Kit+CD34+CD16/32– (CMPs), Lin–Sca-1–c-Kit+CD34+CD16/32+ (GMPs), and Lin–Sca-1–c-Kit+CD34–CD16/32– (MEPs). (F) Epag increased the percentage of GMPs in Tet2+/+ but not Tet2–/– grafted mice. (G–I) Percentage of Tet2–/– cells in indicated populations in bone marrow of transplanted mice. PEP mice were lethally irradiated and received 2 million bone marrow cells consisting of 95% Tet2+/+ (PEP, CD45.1) and 5% Tet2–/– (CD45.2) through tail vein injection. Blood was harvested for flow cytometry analysis, CD11b+CD11c–Ly6C+Ly6G– (monocytes), and CD11b+CD11c–Ly6CloLy6G+ (neutrophils). The antibodies used were FITC-CD45.1, PE-CD11c, APC-Ly6C, Apc-Cy7-Ly6G, and PerCP-CD11b. Data are representative of experiments done twice. (C, D, and F–I) Mice were randomly divided into 2 groups and either treated with 50 mg/kg Epag or vehicle (water) by oral gavage. A total of 4 donor mice and 8 recipient mice were used per group in 2 independent experiments. Data are expressed as mean ± SEM of 8 replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and NS (P > 0.05) by 1-way ANOVA using Dunnett’s test (A) and 2-tailed unpaired t test (B–D and F–I).