Targeting the RNA m6A Reader YTHDF2 Selectively Compromises Cancer Stem Cells in Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m⁶A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m⁶A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.

Keywords: TNFR2; YTHDF2; acute myeloid leukemia; hematopoiesis; hematopoietic stem cell; leukemic stem cells; m(6)A modification; mRNA decay.

Graphical abstract

Figure 1

YTHDF2 Is Upregulated in Different AML Subtypes and Is Essential for AML Development (A) YTHDF2 gene expression in control (CTL) and different cytogenetic subgroups of human AML bone marrow samples. Violin plots show the distribution of log₂ expression values. Horizontal line in the boxplots indicates median. CNG, cytologically normal with good prognosis; CNI, cytologically normal with intermediate prognosis; CAO, cytologically abnormal not otherwise specified. (B) Western blot of YTHDF2 in normal human CD34⁺ cells and AML samples (karyotype details are shown in STAR Methods) (left). α-Histone 3 (H3) was used as a loading control. Quantification of YTHDF2 normalized to H3 expression is presented (right). (C) YTHDF2 gene expression in primitive AML cell compartments with (LSC⁺) and without (LSC⁻) leukemic engraftment potential. (D) Control Ythdf2^fl/fl (Ythdf2^CTL) and Ythdf2^fl/fl;Vav-iCre (Ythdf2^CKO) fetal liver (FL) c-Kit⁺ cells were co-transduced with Meis1 and Hoxa9 retroviruses and serially replated. c-Kit⁺ preleukemic cells were transplanted into recipient mice (n = 12–14). (E) A representative histogram showing GFP-YTHDF2 protein expression in Ythdf2^CTL FL LSK cells and the lack of GFP-YTHDF2 expression in Ythdf2^CKO FL LSK cells. (F) Percentage of GFP-positive cells in the 14.5 days post coitum (dpc) FL LSK cell population from FLs of Ythdf2^CTL and Ythdf2^CKO embryos (n = 5). (G) CFC counts at each replating (n = 3). (H) Percentage of CD11b⁺Gr-1⁻, CD11b⁺Gr-1⁺, and c-Kit⁺ cells in the preleukemic cell compartment (n = 4–5). (I) Percentage of CD45.2⁺ leukemic cells in the PB of recipient mice (n = 12–14 per genotype). (J) Survival curve of recipients transplanted with preleukemic cells (n = 12–14). (K) Percentage of GFP-positive cells in the CD45.2⁺ cell population from moribund recipients of Ythdf2^CTL and Ythdf2^CKO cells (n = 5–6). (L) Limiting dilution assay (LDA). Secondary recipients (n = 5–8) were transplanted with indicated doses of CD45.2⁺ BM cells from primary recipients. (M) Ythdf2^CTL and Ythdf2^CKO FL c-Kit⁺ cells were transduced with MOZ-TIF2 or PML-RARA retroviruses and serially replated. CFC counts at each replating are shown (n = 3). Data represent mean ± SEM; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001.

Figure 2

Loss of YTHDF2 from Established LSCs and Human AML Cells Compromises Their Ability to Propagate AML (A) Ythdf2^fl/fl (Ythdf2^CTL) and Ythdf2^fl/fl;Mx1-Cre (Ythdf2^iCKO) FL c-Kit⁺ cells were co-transduced with Meis1 and Hoxa9 retroviruses, serially replated, and transplanted into primary recipients. GFP⁺c-Kit⁺CD45.2⁺ cells sorted from leukemic primary recipients were re-transplanted into secondary recipients (n = 14–16). (B) Percentage of GFP-expressing cells as a measure of YTHDF2 expression in Ythdf2^CTL and Ythdf2^iCKO leukemic cells prior to secondary transplantation. (C) Percentage of CD45.2⁺ leukemic cells in the PB of the secondary recipient mice 3 weeks after transplantation (n = 14–16 recipients). (D) Survival curve of mice transplanted with Ythdf2^CTL and Ythdf2^iCKO leukemic cells (n = 14–16 mice). (E) Percentage of GFP-expressing cells in PB CD45.2⁺ cell compartment of secondary recipient mice. (F) Left: relative levels of YTHDF2 mRNA (normalized to HPRT1) in human AML THP-1 cells transduced with lentiviruses expressing scrambled short hairpin RNA (shRNA) (CTL) and two independent shRNAs targeting YTHDF2 (KD1 and KD2); n = 3. Right: western blot of YTHDF2 in THP-1 cells shown on the left. α-Histone 3 (H3) was used as a loading control. (G) Proliferation assays with THP-1 cells with CTL, KD1, and KD2 shRNAs. (H) Percentage of Annexin V⁺DAPI⁻ cells. (I) Percentage of CD11b⁻CD14⁻, CD11b⁺CD14⁻, CD11b⁺CD14⁺, and CD11b⁻CD14⁺ cells in cultures shown in (G) and (H). (J) NSG mice were injected with THP-1 cells transduced with CTL (n = 4) or KD (n = 4) lentiviruses and analyzed 1 month later. Percentage of human CD45⁺CD33⁺ cells in the BM, liver, spleen, and PB of the recipient mice is shown. (K) Survival curve of mice transplanted with 10,000 (n = 6) and 1,000 (n = 3) THP-1 cells. (L) Three independent human primary AML samples (AML1–AML3; detailed in STAR Methods) were transduced with CTL, KD1, and KD2 lentiviruses. The graph shows AML-CFC frequencies after 7 days of culture (n = 3 technical replicates per sample). (M) Representative colony images from (L). Data represent mean ± SEM in (A)–(K) or mean ± SD in (L)–(M); ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 3

Ythdf2 Deletion Results in HSC and Progenitor Cell Expansion and Enhanced HSC Reconstitution Potential (A) GFP expression in the BM cell populations from 8- to 12-week-old Ythdf2^fl/fl (Ythdf2^CTL) mice. YTHDF2 is uniformly expressed in BM Lin⁻Sca-1⁺c-Kit⁺ (LSK) cells, LSKCD48⁻CD150⁺ HSCs, LSKCD48⁻CD150⁻ multipotent progenitors (MPPs), primitive hematopoietic progenitor cells (i.e., LSKCD48⁺CD150⁻ HPC-1 and LSKCD48⁺CD150⁺ HPC-2 populations), and Lin⁻Sca-1⁻c-Kit⁺ (LK) myeloid progenitors, and its expression is decreased in differentiated Lin⁺ cells. Data represent mean fluorescence intensity (MFI) ± SEM (n = 4). (B) PB counts of Ythdf2^CTL and Ythdf2^CKO in 8- to 10-wk-old mice (n = 8–9). (C) CFU assays performed with BM cells from 8- to 10-wk-old mice. CFU-Red, CFU-erythroid and/or megakaryocyte; CFU-G, CFU-granulocyte; CFU-M, CFU-monocyte/macrophage; CFU-GM, CFU–granulocyte and monocyte/macrophage; CFU-Mix, at least three of the following: granulocyte, erythroid, monocyte/macrophage, and megakaryocyte (n = 4). (D) FACS profiles showing frequencies (± SEM) of BM LSK, HSC, MPP, HPC-1, and HPC-2 cell populations from Ythdf2^CTL and Ythdf2^CKO mice (n = 6–7 mice). (E) Total number of BM cell populations presented in (D). (F) Ythdf2^fl/fl;Mx1-Cre (Ythdf2^iCKO) and control Ythdf2^fl/fl (Ythdf2^CTL) mice were injected with pIpC and analyzed 3 months after the last injection. (G) Graph showing the percentage of GFP-positive cells in BM of pIpC-treated Ythdf2^iCKO and Ythdf2^CTL mice (n = 10–12). (H) Total BM cellularity of pIpC-treated Ythdf2^iCKO and Ythdf2^CTL mice. (I) Total cell numbers of BM monocytes, granulocytes, and B cells. (J) Total cell numbers of BM LSK and LK cell populations. (K) HSCs were transplanted into lethally irradiated recipient mice (n = 6–9) together with competitor BM cells. Graph shows the percentage of CD45.2⁺ cells overall in the PB and in the monocyte, granulocyte, B cell, and T cell compartments of the PB of primary recipients. (L and M) Percentage of CD45.2⁺ cells in the Lin⁺, Lin⁻, LK, LSK, and HSC (L) and differentiated (M) cell compartments in the BM of recipient mice. Data represent mean ± SEM; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 4

YTHDF2 Targets m⁶A-Methylated Transcripts for Degradation (A) Transcript expression scatterplot from Ythdf2^CTL and Ythdf2^CKO preleukemic cells (n = 5). Significantly upregulated or downregulated transcripts are highlighted in red (p < 0.05). (B) m⁶A peak false discovery rate (FDR) (−log₁₀Q) in Ythdf2^CTL preleukemic cells for transcripts grouped according to expression changes between Ythdf2^CTL and Ythdf2^CKO preleukemic cells is shown (down, genes significantly downregulated in Ythdf2^CKO [p < 0.05]; unchanged, genes not significantly changing in Ythdf2^CKO; up, genes significantly upregulated in Ythdf2^CKO [p < 0.05]). The upper and lower quartiles and the median are shown for each group. (C) Violin plots showing expression change between Ythdf2^CTL and Ythdf2^CKO preleukemic cells for not-methylated (no m⁶A), methylated (m⁶A, −log₁₀Q ≤ 25), and highly methylated (m⁶A high, −log₁₀Q > 25) transcripts. The upper and lower quartiles and the median are indicated for each group. (D) Cumulative distributions of transcript expression change in Ythdf2^CTL and Ythdf2^CKO preleukemic cells for not-methylated, methylated, and highly methylated transcripts as in (C). (E) Mode decay curves for Ythdf2^CTL (black) and Ythdf2^CKO (red) preleukemic cell transcriptomes are shown. The shaded areas indicate the first and third quantile decay curves range for each genotype. Transcript half-life modes for each genotype are indicated with horizontal dotted lines and are also shown at the panel top. (F) Cumulative distributions of transcript half-life in Ythdf2^CTL (left) and Ythdf2^CKO (right) preleukemic cells are shown for not methylated, methylated and highly methylated transcripts as in (C). The half-life change significance between methylated and not-methylated transcripts is indicated. (G) Cumulative distributions of relative stability change between Ythdf2^CTL and Ythdf2^CKO preleukemic cells are shown for not-methylated, methylated, and highly methylated transcripts as in (C). The relative stability change significances between the methylated and not methylated transcripts are indicated. (H) Volcano plot of translational efficiency change between Ythdf2^CTL and Ythdf2^CKO preleukemic cells. Not-methylated, methylated, and highly methylated transcripts defined as in (C) are shown in black, green, and red, respectively. (I) Cumulative distributions of translational efficiency of not-methylated (right), methylated (middle), and highly methylated transcripts (left) defined as in (C) are shown for Ythdf2^CTL (black) and Ythdf2^CKO (red) preleukemic cells. (J) Violin plots of m⁶A peak FDR (−log₁₀Q) in MA9.3ITD and NOMO-1 cells for transcripts grouped according to expression changes between Ythdf2^CTL and Ythdf2^CKO preleukemic cells as in (B) are shown. The upper and lower quartiles and the median are indicated for each group. (K) CPDB analysis of genes significantly upregulated in Ythdf2^CKO preleukemic cells (p < 0.05) with high m⁶A levels (−log₁₀Q > 25) in mouse preleukemic cells and also methylated in human AML cell lines. (L) GSEA using LSC signature gene set for genes defined in (K) and that negatively correlate with YTHDF2 expression in human AML samples. (M) m⁶A immunoprecipitation (IP) read coverage (blue) from Ythdf2^CTL preleukemic cells along the Trnfrs1b genomic locus (top) and m⁶A IP read coverage from NOMO-1, and MA9.3ITD cells along the TNFRSF1B genomic locus (bottom) are shown. Input coverage is shown in green. (N) Tnfrsf1b enrichment in YTHDF2 immunoprecipitates from Ythdf2^CTL preleukemic cells is shown. Tnfrsf1b background levels were determined using Ythdf2^CKO preleukemic cells (n = 3). (O) Decay curves for Trnfrs1b in Ythdf2^CTL (top) and Ythdf2^CKO (bottom) preleukemic cells transcriptomes are shown. The center value and the error bars at each time point indicate the conversion rate mean and SD, respectively. The conversion rates for each biological replicate are indicated with dots. The Trnfrs1b half-life is also shown. (P) FACS plots showing the expression of TNFR2 on the cell surface of Ythdf2^CTL and Ythdf2^CKO preleukemic cells. The inner graph displays the quantification of TNFR2 expression (n = 4). (Q) Percentage of Annexin V⁺DAPI⁻ preleukemic cells treated with TNF-α at 0-h and 6-h time points (n = 3). Data in (N), (P), and (Q) represent mean ± SEM. In (B), (C), (J), (N), (P), and (Q) ^∗p < 0.05; ^∗∗p < 0.01.