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. 2018 Nov 29;132(22):2406-2417.
doi: 10.1182/blood-2018-05-853291. Epub 2018 Sep 25.

TET2 deficiency leads to stem cell factor-dependent clonal expansion of dysfunctional erythroid progenitors

Xiaoli Qu  1   2 , Shijie Zhang  1 , Shihui Wang  1 , Yaomei Wang  1   2 , Wei Li  1   2   3 , Yumin Huang  2   4 , Huizhi Zhao  1 , Xiuyun Wu  1 , Chao An  2   4 , Xinhua Guo  2 , John Hale  5 , Jie Li  1   2 , Christopher D Hillyer  5 , Narla Mohandas  5 , Jing Liu  6 , Karina Yazdanbakhsh  7 , Francesca Vinchi  8 , Lixiang Chen  1 , Qiaozhen Kang  1 , Xiuli An  1   2
Affiliations

TET2 deficiency leads to stem cell factor-dependent clonal expansion of dysfunctional erythroid progenitors

Xiaoli Qu et al. Blood. .

Abstract

Myelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Anemia is the defining cytopenia of MDS patients, yet the molecular mechanisms for dyserythropoiesis in MDSs remain to be fully defined. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is 1 of the most common mutations in MDSs and that TET2 deficiency disturbs erythroid differentiation. However, mechanistic insights into the role of TET2 on disordered erythropoiesis are not fully defined. Here, we show that TET2 deficiency leads initially to stem cell factor (SCF)-dependent hyperproliferation and impaired differentiation of human colony-forming unit-erythroid (CFU-E) cells, which were reversed by a c-Kit inhibitor. We further show that this was due to increased phosphorylation of c-Kit accompanied by decreased expression of phosphatase SHP-1, a negative regulator of c-Kit. At later stages, TET2 deficiency led to an accumulation of a progenitor population, which expressed surface markers characteristic of normal CFU-E cells but were functionally different. In contrast to normal CFU-E cells that require only erythropoietin (EPO) for proliferation, these abnormal progenitors required SCF and EPO and exhibited impaired differentiation. We termed this population of progenitors "marker CFU-E" cells. We further show that AXL expression was increased in marker CFU-E cells and that the increased AXL expression led to increased activation of AKT and ERK. Moreover, the altered proliferation and differentiation of marker CFU-E cells were partially rescued by an AXL inhibitor. Our findings document an important role for TET2 in erythropoiesis and have uncovered previously unknown mechanisms by which deficiency of TET2 contributes to ineffective erythropoiesis.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Hyperproliferation of TET2-knockdown CFU-E cells in the presence of SCF. (A) qRT‐PCR analyses of TET2 mRNA levels (normalized to actin) of sorted luciferase and TET2-knockdown erythroid cells. (B) Colony-forming ability of sorted luciferase and TET2-knockdown CFU-E cells in the presence of EPO only (left panel) or in the presence of EPO plus SCF (right panel). Scale bar, 50 μm. (C) Percentage of larger-size colonies in panel B. (D) Growth curves of sorted luciferase and TET2-knockdown CFU-E cells cultured in the presence of EPO plus SCF. (E) Numbers of cell divisions of sorted erythroblasts at the indicated stages in culture. All results are from 3 independent experiments. *P < .05, **P < .01, ***P < .001.
Figure 2.
Figure 2.
TET2 knockdown impaired human terminal erythroid differentiation. (A) Representative flow cytometry analysis of GPA expression of sorted luciferase and TET2-knockdown CFU-E cells cultured in the presence of EPO plus SCF for different days, as indicated. (B) Quantitative analysis of changes in GPA− population from 3 independent experiments. (C) Terminal erythroid differentiation was monitored on the indicated days by flow cytometric analysis based on the expression of band 3 and α4 integrin. Representative plots of α4-integrin vs band 3 of GPA+ cells are shown, and the erythroblasts are separated into 5 populations: proerythroblasts (Pro; α4-integrinhiband 3neg), early basophilic erythroblasts (EB; α4-integrinhiband 3low), late basophilic erythroblasts (LB; α4-integrinhiband 3med), polychromatic erythroblasts (Poly; α4-integrinmedband 3med), and orthochromatic erythroblasts (Ortho; α4-integrinlowband 3hi). (D) Quantitative analyses of results shown in panel C from 3 independent experiments. (E) Representative cytospin images of erythroblasts cultured for different days as indicated. *P < .05, **P < .01.
Figure 3.
Figure 3.
TET2 knockdown led to upregulation and activation of c-Kit. (A) mRNA levels (normalized to actin) of c-Kit, as assessed by qRT-PCR. (B) Representative western blot analysis of total c-Kit, p–c-Kit, and SHP-1. GAPDH was used as loading control. (C) Quantitative analysis of c-Kit, p–c-Kit, and SHP-1 from 3 independent experiments. (D) Effects of c-Kit inhibitor STI571 (0.5 μM) on proliferation of sorted luciferase and TET2-knockdown CFU-E cells. Dimethyl sulfoxide (DMSO) was used as control. (E-H) Effects of c-Kit inhibitor STI571 (0.5 μM) on differentiation of luciferase and TET2-knockdown CFU-E cells. Sorted luciferase and TET2-knockdown CFU-E cells were cultured for 13 days in the presence of DMSO or STI571. Expression of GPA (E) or band 3/α4 integrin (F) was examined by flow cytometry analysis. (G) Quantitative analysis of results shown in panel F from 3 independent experiments. (H) Representative cytospin images of erythroblasts. Note that the TET2-knockdown–induced impairment in differentiation was reversed by STI571 treatment. *P < .05, **P < .01, ***P < .001.
Figure 4.
Figure 4.
Knockdown of TET2 led to the generation of marker CFU-E cells. (A) TET2-knockdown CFU-E cells were cultured for 13 days, and the GPA− population was sorted. (B) Representative cytospin images of sorted luciferase CFU-E and TET2-knockdown GPA− cells. (C) Flow cytometry analysis of luciferase CFU-E and TET2-knockdown GPA− cells using the surface markers IL-3R, GPA, CD34, CD36, and CD71. (D) Proliferation of luciferase CFU-E and TET2-knockdown GPA− cells in the presence of EPO only or EPO plus SCF. (E-F) Differentiation of luciferase CFU-E and TET2-knockdown GPA− cells. Sorted luciferase and TET2-knockdown GPA− cells were cultured for 5 or 15 days. Expression of GPA (E) or band 3/α4 integrin (F) was examined by flow cytometry analysis. (G) Quantitative analysis of results shown in panel F from 3 independent experiments. (H) Representative cytospin images of erythroblasts. (I) Western blot analysis of c-Kit, p–c-Kit, AKT, p-AKT, ERK, p-ERK, and SHP-1. GAPDH was used as loading control. (J) Quantitative analysis of results shown in panel H from 3 independent experiments. *P < .05, **P < .01, ***P < .001.
Figure 5.
Figure 5.
Upregulation of AXL in marker CFU-E cells. (A) Number of differentially expressed genes among luciferase CFU-E, TET2-knockdown CFU-E, and TET2-knockdown marker CFU-E cells, as revealed by RNA-seq analysis. Fold change > 2. (B) Heat map of the 6 genes involved in the “organ regeneration” pathway. (C) mRNA levels of AXL, as revealed by RNA-seq. (D) mRNA levels of AXL, as assessed by qRT-PCR. (E) Western blot analysis of AXL. (F) Quantitative analysis of AXL protein levels from 3 independent experiments. (G) Growth curves of sorted luciferase and TET2-knockdown CFU-E cells in the presence of DMSO or 0.2 μM R428. (H) Flow cytometry analysis showing GPA expression of luciferase and TET2-knockdown CFU-E cells cultured for 13 days in the presence of DMSO or 0.2 μM R428. (I) Flow cytometry analysis showing band 3/α4 integrin expression of luciferase and TET2-knockdown CFU-E cells cultured for 13 days in the presence of DMSO or 0.2 μM R428. (J) Quantitative analysis of results shown in panel I. (K) Representative cytospin images of erythroblasts. *P < .05, **P < .01, ***P < .001.
Figure 6.
Figure 6.
Effects of TET2 knockdown on DNA methylation. (A) DNA dot blots for 5mC. (B) DNA dot blots for 5hmC. (C) Quantitative analysis of 5mC and 5hmC levels from 3 independent experiments. (D) AXL promoter region containing 6 CCGG sites (blue). (E-F) Methylation-sensitive restriction enzyme analysis of 5mC and 5hmC changes at −414, +320 CCGG sites of AXL promoter. *P < .05.
Figure 7.
Figure 7.
TET2 knockdown led to the SCF-dependent clonal expansion of CFU-E cells. (A) Growth of sorted single luciferase or TET2-knockdown CFU-E cells cultured in the presence of EPO for 7 days. (B) Growth of sorted single luciferase or TET2-knockdown CFU-E cells cultured in the presence of EPO plus SCF for 15 days. Note that cells can be divided into HP and LP populations. (C) Representative cytospin images of erythroblasts from panel B. (D) mRNA levels of AXL in the LP cells (pooled) and HP cells (pooled). (E) Western blot analysis of AXL of pooled LP and HP cells. (F) Quantitative analysis of AXL protein levels from 3 independent experiments. *P < .05. NS, not statistically significant.
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