TET2-mediated mRNA demethylation regulates leukemia stem cell homing and self-renewal

Abstract

TET2 is recurrently mutated in acute myeloid leukemia (AML) and its deficiency promotes leukemogenesis (driven by aggressive oncogenic mutations) and enhances leukemia stem cell (LSC) self-renewal. However, the underlying cellular/molecular mechanisms have yet to be fully understood. Here, we show that Tet2 deficiency significantly facilitates leukemogenesis in various AML models (mediated by aggressive or less aggressive mutations) through promoting homing of LSCs into bone marrow (BM) niche to increase their self-renewal/proliferation. TET2 deficiency in AML blast cells increases expression of Tetraspanin 13 (TSPAN13) and thereby activates the CXCR4/CXCL12 signaling, leading to increased homing/migration of LSCs into BM niche. Mechanistically, TET2 deficiency results in the accumulation of methyl-5-cytosine (m⁵C) modification in TSPAN13 mRNA; YBX1 specifically recognizes the m⁵C modification and increases the stability and expression of TSPAN13 transcripts. Collectively, our studies reveal the functional importance of TET2 in leukemogenesis, leukemic blast cell migration/homing, and LSC self-renewal as an mRNA m⁵C demethylase.

Keywords: CXCR4; RNA 5-methylcytosine methylation; TET2; TSPAN13; YBX1; bone marrow microenvironment; homing; leukemia stem cell; migration; self-renewal.

Figure 1.. TET2 deficiency predicts poor prognosis in AML patients and promotes primary leukemogenesis.

(A) Kaplan-Meier survival curves of TCGA AML patients TET2 high (n=44; roughly the top 1/4) and low/medium (n=119; the remaining patients) expression. The X-tile software was used to determine the optimal cutoff values for predicting survival. (B) Violin plots showing the expression of TET2 in healthy donors and AML patients. The expression values, derived from Affymetrix exon arrays, were log₂ transformed. (C) Violin plots showing the relative expression of TET2 in favorable and adverse AML patients in the Beat AML cohort. (D) Kaplan-Meier survival curves of AML patients with or without TET2 mutation. WT, wild-type (without TET2 mutation in the Beat AML cohort, http://vizome.org/aml/). n = 54 for TET2 mutation group; n = 415 for TET2 WT group. (E) Schematic outline of primary BMT models driven by various gene mutations and translocations. (F-H) Kaplan-Meier survival curves of NRAS^mut & AE9a (F), NRAS^mut & PML-RARA (G), and DNMT3A^mut & NRAS^mut (H)-driven primary murine AML models upon Tet2 deficiency. Tet2 (ΔE8–10) strain was used for F and G; Tet2 (ΔE3) strain was used for H. (I-K) The white blood cell (WBC) count (I), red blood cell (RBC) count (J), and platelet (PLT) count (K) in the recipient mice transplanted with Tet2 ^+/+ versus Tet2 ^−/− (ΔE8–10) primary NRAS/AE9a (N&A) or NRAS/PML-RARA (N&P) cells (n = 4). (L) The spleen weight and representative spleens of DNMT3A^mut & NRAS^mut -driven primary murine AML models (n = 3). (M and N) The WBC count in the recipient mice transplanted with primary DNMT3A^mut & NRAS^mut (M)- or FLT3^ITD (N)-transformed Tet^+/+ or Tet2^−/− mouse BM progenitor cells (n = 3). (O) Effect of Tet2 deficiency on the engraftment of FLT3^ITD leukemia cells into peripheral blood (PB), spleen (SP), and BM of primary recipient mice. The samples were collected on day 52 post transplantation (n = 3). (P) The representative spleens of FLT3^ITD-driven primary murine AML models. The samples were collected on day 52 post transplantation. Unpaired two-tailed Student’s t-test (B, C, and I-O); log-rank test (A, D, and F-H); *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1.

Figure 2.. Tet2 depletion significantly enhances LSC self-renewal while its overexpression attenuates LSC self-renewal and AML progression in various models.

(A) Enrichment plot of Ivanova hematopoiesis stem cell pathway which was activated in Tet2^−/− (ΔE8–10) primary NRAS/AE9a AML cells. (B) IGV tracks of Tet2 in Tet2 ^+/+ or Tet2 ^−/− (ΔE8–10) NRAS/AE9a AML cells. (C) Colony numbers from serial replating assays with Tet2 ^+/+ and Tet2 ^−/− (ΔE3) primary leukemia cells. The representative data from NRAS/AE9a model was shown. (D) FACS plots depicting the percentage of LK (Lin⁻c-Kit⁺Sca-1⁻) and LSK (Lin⁻c-Kit⁺Sca-1⁺) subpopulations in Tet2 ^+/+ and Tet2 ^−/− (ΔE3) NRAS/AE9a AML cells. (E) The representative colony images of Tet2 ^+/+ or Tet2 ^−/− (ΔE3) primary AML cells driven by FLT3^ITD, DNMT3A^mut & NRAS^mut, and DNMT3A^mut & FLT3^ITD. (F) Effect of Tet2 depletion on the colony numbers of primary AML cells driven by FLT3^ITD (left), DNMT3A^mut & NRAS^mut (middle), and DNMT3A^mut & FLT3^ITD (right). (G) Kaplan-Meier survival curves of secondary BMT recipient mice. The leukemic BM cells isolated from the primary BMT recipient mice of Tet2^+/+ + MA9 or Tet2^−/− (ΔE8–10) + MA9 were used as donor cells. (H) Representative spleen images of the secondary BMT models. All the spleens were collected on day 27 post transplantation of Tet2^+/+ + MA9 or Tet2^−/− (ΔE8–10) + MA9 primary AML cells. (I) The in vivo limiting dilution assay with Tet2^+/+ + MA9 or Tet2^−/− (ΔE8–10) + MA9 primary BM leukemia cells. CD45.2 recipients (n = 5 for each group) were transplanted with different doses of leukemia cells and the estimated LSC frequencies were displayed. (J) TET2 protein levels in the CD34⁺ and CD34⁻ populations of primary AML patient samples as determined by intracellular staining. (K) Comparison of mean fluorescence intensity (MFI) of TET2 protein levels between CD34⁺ and CD34⁻ populations in primary AML patient samples. (L-M) Effect of TET2 overexpression on the colony number (L) and cell number (M) of Mono Mac 6 AML cells. (N) Effect of TET2 overexpression on the colony number of AML PDX (PDX-148, carrying DNMT3A mutation) cells. (O) The bioluminescence images of AML PDX model (PDX-129, carrying a complex karyotype) with or without TET2 overexpression. The images were photted on day 25 post transplantation. (P) The statistical bioluminescence signals of AML PDX model with or without TET2 overexpression. (Q) Kaplan-Meier survival curves of AML PDX models with or without TET2 overexpression. (R) Kaplan-Meier survival curves of Mono Mac 6-induced AML xenograft models with or without TET2 overexpression. Unpaired two-tailed Student’s t-test (C, F, L-N, and P); log-rank test (G, Q, and R); paired two-tailed Student’s t-test (K); *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S2 and S3.

Figure 3.. Tet2 deficiency promotes the migration of AML blast cells or LSCs into surrounding stroma to maintain their self-renewal.

(A-B) Effect of Tet2 depletion on the proliferation of primary MA9 cells in single culture condition (A) and stroma cell (OP-9) co-culture condition (B). The numbers of days after culture or co-culture are indicated under the x-axis. (C-D) The effect of Tet2 deficiency on the proliferation of MA9-transformed leukemic cells after co-culturing with OP9 stromal cells for 8 days, as determined by 5-ethynyl-2′-deoxyuridine (EdU) staining. C, the statistical results of the percentage of EdU positive cells; D, representative flow cytometry data. (E) Scheme showing the single culture and co-culture of primary AML cells with OP-9 stromal cells before colony-forming assay (left) as well as the representative colony images (right). The AML cells were co-cultured with OP-9 cells for 5 days before colony-forming assay. For each group, 150 AML cells were seeded into 96-well plate. (F-G) The colony number (F) and LSC frequency (G, in vitro LDA) of Tet2^+/+ and Tet2^−/− primary MA9 AML cells with or without co-culture. (H) Scheme showing the transwell assay to evaluate the migration ability of Tet2^+/+ and Tet2^−/− primary AML cells. (I-K) The effect of Tet2 deficiency on the migration ability of primary AML cells carrying MA9 (I), FLT3^ITD (J), and DNMT3A & NRAS mutations (K). (L) Effect of TET2 KO on the migration ability of THP1 AML cells. Unpaired two-tailed Student’s t-test (A-C, F, and I-L); **p < 0.01, ***p < 0.001. See also Figures S4.

Figure 4.. Te2 depletion facilitates LSC homing in vivo.

(A) Schematic outline of the method to evaluate LSC homing in vivo. (B) The in situ immunofluorescence (IF) of MA9 primary leukemia cells (CFSE-labeled; Green) and BM osteoblasts (Osteopontin, OPN; Red) in the femurs from Tet2^+/+ and Tet2^−/− (ΔE8–10) group. While arrows indicated CFSE-labeled leukemia cells. The samples were collected at 16 hours post transplantation. (C) Quantitative and statistical analysis of in situ IF images in Figure 4B. (D) Effect of Tet2 depletion on the homing of primary MA9 AML cells into the BM of recipient mice. The data were determined by flow cytometry. The samples were collected at 16 hours post transplantation. (E) Comparison of the percentage between Tet2^−/− and Tet2^+/+ primary MA9 AML cells homing into BM of recipient mice via flow cytometry. The samples were collected at 1, 4, 12, and 16 hours post transplantation. (F) Effect of Tet2 depletion on the homing of primary NRAS/AE9a AML cells. The data were determined by flow cytometry. The samples were collected at 16 hours post transplantation. (G) Evaluation of effect of Tet2 depletion on homing via secondary MA9 BMT models. The samples were collected at 16 hours post transplantation. (H) Effect of TET2 overexpression on the homing of AML PDX cells into the BM of NRGS recipient mice. The samples were collected at 16 hours post transplantation. For C-H, data were presented as mean ± SEM (n = 3). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed Student’s t test. See also Figures S5.

Figure 5.. Identification of Tspan13 as a functional essential target of Tet2.

(A) Overlap of core enriched genes in Ivanova hematopoiesis stem cell pathway from two Tet2 KO (Tet2^−/− (ΔE8–10) and Tet2^−/− (ΔE3)) NRAS/AE9a AML models. The two Tet2^−/− NRAS/AE9a (i.e., Tet2^−/− (ΔE8–10) NRAS/AE9a and Tet2^−/− (ΔE3) NRAS/AE9a) AML models as well as their corresponding control cells (Tet2^+/+) were subjected to RNA-seq. The left circle (green) indicates the core-enriched genes in the Ivanova hematopoiesis stem cell pathway when comparing Tet2^−/− (ΔE8–10) NRAS/AE9a AML cells with their control cells; while the right circle (red) indicates the core-enriched genes in the same pathway when comparing Tet2^−/− (ΔE3) NRAS/AE9a AML cells with their control cells. (B) Heatmap of expression level changes of the 41 overlapped core enriched genes in Ivanova hematopoiesis stem cell pathway upon Tet2 depletion (ΔE8–10) in the NRAS/AE9a AML model. (C) Bubble plot depicting the correlation between TET2 expression and the 41 overlapped genes in 3 human AML datasets. (D and E) Expression levels of TSPAN13 (D) and SV2A (E) in the favorable and adverse AML patients. The raw data were derived from the Beat AML cohort (http://vizome.org/aml/). The three lines inside the violin plots are the first quartile, median and third quartile; unpaired two-tailed Student’s t-test. (F) IGV track showing the abundance of Tspan13 in Tet2^+/+ or Tet2^−/− (ΔE3) NRAS/AE9a primary leukemia cells. (G) The knockdown (KD) efficiency of Tspan13 in Tet2^−/− (ΔE8–10) MA9 primary leukemia cells as determined by qPCR. (H) The effect of Tsapn13 KD on the colony-forming ability of Tet2^−/− (ΔE8–10) MA9 primary leukemia cells. (I) The representative colony images of Tet2^−/− (ΔE8–10) MA9 primary leukemia cells upon Tspan13 KD. (J) The effect of Tsapn13 KD on the colony-forming ability of Tet2^−/− (ΔE3) NRAS/AE9a primary leukemia cells. (K) The rescued effect of Tspan13 KD on Tet2 deficiency-induced homing. The in vivo homing was determined by flow cytometry. (L) The rescued effect of Tspan13 KD on Tet2 deficiency (ΔE3)-induced increase of LSC frequency as determined by LDA with NRAS/AE9a primary leukemia cells. (M) Survival curves of recipient mice transplanted with Tet2^−/− (ΔE8–10) MA9 primary leukemia cells with or without Tspan13 KD. n = 5 mice per group. The p values were calculated with a log-rank test (n=5). (N) The effect of Tspan13 KD on the engraftment of Tet2^−/− MA9 cells into the BM and Spleen of recipient mice. For G, H, J, K, and N, data were presented as mean ± SEM (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; unpaired two-tailed Student’s t-test. See also Figure S6.

Figure 6.. Tet2 depletion promotes LSC homing via activating the Tspan13/Cxcr4/Cxcl12 signaling.

(A) Protein level changes of Tspan13, p-Cxcr4, and Cxcr4 in primary AML cells upon Tet2 depletion. (B) Protein level changes of TET2-CD-Flag, p-CXCR4, and CXCR4 in AML PDX cells and cell line (Mono Mac 6, MMC6) upon TET2 overexpression. (C) Effect of Tet2 depletion on the expression levels of surface p-Cxcr4 in primary AE9a/NRAS AML cells. (D) Effect of Tspan13 KD on the expression levels of surface p-Cxcr4 in primary Tet2^−/− MA9 AML cells. (E) Schematic outline of transwell assay with stroma cells which were pre-treated with Cxcl12 antibody. The OP9 cells were pretreated with 1μg/ml Cxcl12 antibody for 24–48 hours before co-culture with AML cells. (F) Effect of Cxcl12 antibody on the migration ability of Tet2^−/− MA9 primary AML cells. (G) Effect of Tspan13 KD and Cxcl12 antibody treatment on the migration ability of Tet2^−/− MA9 primary AML cells. (H) Schematic outline of a strategy to evaluate the effect of Cxcr4 inhibitor (Cxcr4i, AMD3100) on the homing. Before transplantation, the MA9 primary AML cells were treated with 0.1mg/ml AMD3100 for 20 hours. (I-J) Statistical results (I) and representative flow cytometry (J) showing the effect of Cxcr4 inhibition on the homing of Tet2^−/− primary AML cells. For C, D, F, G, and I, data were presented as mean ± SEM (n = 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed Student’s t-test.

Figure 7.. Tet2 induces the degradation of Tspan13 and thus negatively regulates its expression as an mRNA m⁵C demethylase.

(A) The possible mechanisms through which Tet2 regulates Tspan13 expression. (B) The transcription efficiency of Tspan13 in Tet2^+/+ and Tet2^−/− (ΔE3) NRAS/AE9a leukemia cells as determined by nuclear run-on assay. (C) Effect of Tet2 deficiency (ΔE3) on Tspan13 mRNA stability in primary NRAS/AE9a AML cells. (D) The calibration curves of cytosine (C), m⁵C, and hm⁵C standards as determined by QQQ-MS. (E) Scheme of the in vitro transcription assay as well as the in vitro (cell-free) demethylation assay with TET2 protein and m⁵C-modified TSPAN13 mRNA. (F and G) Assessing the m⁵C (left) and hm⁵C (right) level changes in TSPAN13 mRNA in the in vitro demethylation assay with TET2 protein as detected by QQQ-MS (F) or dot blot assays (G). (H and I) The abundance of m⁵C in the poly(A) RNA isolated from NRAS/AE9a (H) and MA9 (I) primary leukemia cells with or without Tet2 deficiency. (J) Relative levels of Tsapn13 mRNA m⁵C modification in Tet2^+/+ and Tet2^−/− (ΔE3) NRAS/AE9a primary leukemia cells as determined by gene-specific m⁵C qPCR. (K) The direct interaction between Tet2 protein and TSPAN13 mRNA as detected by CLIP-qPCR in C1498 AML cells. Here, we overexpressed Flag-tagged Tet2 catalytic domain (CD) and performed CLIP with Flag antibody. (L) The direct interaction between Ybx1 (Flag-tagged) and Tspan13 mRNA as detected by CLIP-qPCR in C1498 AML cells. (M) The effect of Ybx1 KD on Tspan13 mRNA stability in C1498 AML cells. (N) The cumulative distribution function (CDF) plot showing the effect of Tet2 depletion on global mRNA stabilities in NRAS/AE9a primary leukemia cells. (O) The MA plot showing the mRNAs with significantly increased stability (Half-life UP; Red) and decreased stability (Half-life Down; Green) in NRAS/AE9a primary leukemia cells upon Tet2 depletion. (P) The schematic diagram showing how Tet2 regulates LSC homing and self-renewal via targeting the m⁵C/TSPAN13/CXCR4 axis in the BM microenvironment. For C, F, and H-N, data were presented as mean ± SEM (n=3 independent experiments). **p < 0.01, ***p < 0.001; unpaired two-tailed Student’s t-test. See also Figure S7.