RNA Demethylase ALKBH5 Selectively Promotes Tumorigenesis and Cancer Stem Cell Self-Renewal in Acute Myeloid Leukemia

Abstract

N⁶-methyladenosine (m⁶A), the most abundant internal modification in mRNA, has been implicated in tumorigenesis. As an m⁶A demethylase, ALKBH5 has been shown to promote the development of breast cancer and brain tumors. However, in acute myeloid leukemia (AML), ALKBH5 was reported to be frequently deleted, implying a tumor-suppressor role. Here, we show that ALKBH5 deletion is rare in human AML; instead, ALKBH5 is aberrantly overexpressed in AML. Moreover, its increased expression correlates with poor prognosis in AML patients. We demonstrate that ALKBH5 is required for the development and maintenance of AML and self-renewal of leukemia stem/initiating cells (LSCs/LICs) but not essential for normal hematopoiesis. Mechanistically, ALKBH5 exerts tumor-promoting effects in AML by post-transcriptional regulation of its critical targets such as TACC3, a prognosis-associated oncogene in various cancers. Collectively, our findings reveal crucial functions of ALKBH5 in leukemogenesis and LSC/LIC self-renewal/maintenance and highlight the therapeutic potential of targeting the ALKBH5/m⁶A axis.

Keywords: ALKBH5; MYC; P21; TACC3; acute myeloid leukemia; hematopoietic stem cells (HSCs); leukemia stem cells (LSCs/LICs); m(6)A modification; normal hematopoiesis; prognosis.

Figure 1.. Biological effects of forced expression or knockdown of ALKBH5 on human AML cells

(A) Comparison of the expression levels of ALKBH5 in primary AML patients bearing various chromosomal translocations with those in BM hematopoietic stem cells (HSCs) collected from healthy donors (NC) based on the GSE13159 and GSE42519 datasets. The expression values (detected by Affymetrix exon arrays) were log2-transformed. (B) Western blot of ALKBH5 protein in normal controls (human bone marrow mononuclear cells (MNCs)) as well as in TP53-wild-type and -mutant AML cell lines. ACTIN was used as a loading control. (C) Kaplan-Meier survival analysis in TCGA-AML dataset (n=106). The patients were divided into two groups of equal size based on ALKBH5 levels. The p value was detected by the log-rank test. (D and E) Cell growth/proliferation assays in MONOMAC6 (MMC6) (D) and NOMO1 (E) AML cell lines (TP53-mutant) transduced with lentiviruses expressing control shRNA (shNS) or two independent shRNAs targeting ALKBH5 (shA5-#1 and shA5-#2), as well as those expressing empty vector (Vector), wild-type ALKBH5 (A5-WT) and m⁶A demethylase-inactive ALKBH5 mutant (A5-Mut). (F and G) Representative flow cytometry plots (F) and statistics (G) of the percentage of apoptotic cells in MMC6 and NOMO1 cells with shNS or ALKBH5 shRNAs. (H and I) m⁶A dot blot assays (H) and quantitative comparison (I) of global m⁶A abundance in MMC6 and NOMO1 cells with shNS or ALKBH5 shRNAs (n=3 biological replicates). MB, methylene blue staining (as loading control). Mean±SD values are shown for Figures 1A, D, E, G and I. *p < 0.05; **p <0.01; ***p < 0.001; t test. See also Figure S1.

Figure 2.. The role of Alkbh5 in MLL-AF9 (MA9) mediated leukemogenesis.

(A) Scheme of the design and procedures of generation of Alkbh5 knockout (KO) mice using the CRISPR-Cas9 technology. (B and C) Representative DNA genotyping (B) and Western blot assay (C) data of the samples from Alkbh5 wild-type (WT), heterozygous (Heter) or homozygous (Homo) KO mice were shown. (D) Bone marrow (BM) lineage negative (Lin⁻) cells collected from Alkbh5 WT, Heter or Homo KO mice were transduced with MA9 retrovirus and used for colony-forming/replating (CFA) assays. The MA9-transduced cells were also transplanted into lethally irradiated recipient mice after first round of colony formation for leukemogenesis. (E) Colony forming cell counts at each round of plating were shown (n=3). (F) Kaplan-Meier survival curves of recipient mice transplanted with MA9-transduced Alkbh5 WT (n=11), Heter (n=7) and Homo (n=8) HSPCs. (G-M) Three mice from each transplant group were euthanized at the same time (day 61 post bone marrow transplantation (BMT)) for AML development analysis. (G-H) Percentage of CD45.2⁺ cells in peripheral blood (PB) (G) and BM (H) of recipient mice. (I) WBC count in PB of recipient mice. (J) Spleen weight of recipients. (K-L) Percentage of Mac-1⁺c-Kit⁺ cells in recipient mice. Representative flow cytometry plots (K) and statistics analysis (L) are shown. (M) Representative images of Wright-Giemsa staining of BM and PB from recipient mice. *p < 0.05; **p <0.01; ***p < 0.001; t test (for Figures 2E, G–J and L; Mean±SD values are shown) or log-rank test (for Figure 2F). See also Figure S2.

Figure 3.. Knockdown of ALKBH5 affects the maintenance of human and murine AML.

(A) Primary mouse leukemic BM cells were transduced with lentiviruses expressing control shRNA (shNS) and two independent shRNAs targeting Alkbh5 (shA5-#a and shA5-#b) and serially replated. Colony forming cell counts at each round of plating were shown (n=3). (B) Western blot of Alkbh5 (left) and dot blot of global m⁶A abundance (right) in mouse MA9 AML cells transduced with shNS or Alkbh5 shRNAs. Actin was used as a loading control in Western blot. MB, methylene blue staining, was used as loading control in m⁶A dot blot. (C) Kaplan-Meier survival curves of recipients transplanted with mouse MA9 AML cells with shNS or Alkbh5 shRNA (shA5-#b) (n=7 for each group). (D-E) WBC count (D) and percentage of CD45.2⁺ cells (E) in the PB of recipient mice. (F and G) Human AML cells were transduced with shNS or ALKBH5 shRNAs and then plated for colony forming assays. (F) Colony forming cell counts (n=3). (G) Representative pictures of colonies from NOMO1 cells. MA9.3-ITD (P53 wild-type), NOMO1 (P53 mutant) and MV4;11 (P53 wild-type) were used. (H) Kaplan-Meier survival curves of NSGS mice transplanted with MV4;11 AML cells that were transduced with shNS (n=7) or ALKBH5 shRNAs (shA5-#1, n=9; shA5-#2, n=10) (0.1×10⁶ donor cells/mouse). (I-N) Primary leukemia cells from AML patients were transduced with shNS or ALKBH5 shRNAs and then seeded for experiments. (I, L) Cell growth/proliferation assays of transduced primary AML cells. (J, M) Colony forming cell counts of transduced AML cells. (K, N) Percentage of apoptotic cells in overall (top panel) or CD34⁺ (lower panel) transduced AML cells. *p < 0.05; **p <0.01; ***p < 0.001; t test (for Figures 3A, D–F, I–J and L–M; Mean±SD values are shown) or log-rank test (for Figures 3C and H). See also Figure S3.

Figure 4.. Effects of Alkbh5 depletion on self-renewal/repopulation of leukemia stem/initiating cells (LSCs/LICs) and normal hematopoiesis.

(A-C) BM lin- cells from Alkbh5 WT or homozygous KO mice were transduced with MA9-YFP retrovirus and then transplanted into primary recipient mice. Five mice from each transplant group were euthanized at the same time (day 42 post-BMT) for LSC/LIC analysis. (A) Representative flow cytometry analysis of LSCs/LICs in BM. (B) Percentage of leukemic GMPs (LGMPs) in YFP⁺ cells. (C) Percentage of apoptotic LGMPs. (D and E) YFP⁺ BM cells were sorted out from primary BMT mice and transplanted into secondary recipient mice for 2^nd BMT assay. (D) Survival curves of the 2^nd BMT mice (MA9-WT, n=12; MA9-KO, n=10). (E) WBC count from the 2^nd BMT mice euthanized on day 25 post-BMT. (F) The in vivo limiting dilution assay (LDA). Secondary recipients (n=5 for each group) were transplanted with different doses of BM cells collected from primary recipients (see Figure 2F) euthanized at the same time (Day 61 post BMT). (G-N) Effects of Alkbh5 KO on mouse static normal hematopoiesis. (G) Regularly bred 6- to 10-weeks Alkbh5 WT (n=7) and KO (n=10) mice were included for the analysis. PB CBC counts were shown in (H). BM analysis of 4 pairs of the same sex/age littermates were shown in (I-N). (O-R) The in vivo competition assay. (O) Schematic outline of experiment strategy of the in vivo competition assays. (P) Ratio of CD45.2⁺/CD45.1⁺CD45.2⁺ in the PB of recipients. (Q and R) Ratio of CD45.2⁺/CD45.1⁺CD45.2⁺ in the Lin⁺, Lin⁻, LK, LSK, HSC and other progenitor cell populations (Q) and differentiated cell compartments (R) in the BM of recipients. *p < 0.05; **p <0.01; ***p <0.001; t test (for Figures 4B–C, E, H–N, P–R; Mean±SD values are shown) or log-rank test (for Figures 4D and F). See also Figure S4.

Figure 5.. Transcriptome-wide identification of ALKBH5 potential targets in AML cells.

(A-C) RNA-seq analysis of gene expression profiles in ALKBH5 knockdown AML cells and control AML cells. (A) Venn diagram shows numbers of genes with significant changes in expression (RPKM>1, fold change>1.5) upon ALKBH5 knockdown. (B) GSEA of up- and down-regulated genes. (C) Violin plots showing the relative abundance of genes involved in the indicated pathways in ALKBH5 knockdown or control NOMO1 cells. (D-F) RIP-seq analysis of ALKBH5 overexpressing NOMO1 cells. (D) Scatter plots of ALKBH5 RIP-seq replicates showing the correlation of enriched genes. (E) Pie charts showing the distribution of RIP-seq reads in RNA classes. (F) GSEA of significantly enriched genes in RIP samples (RPKM>1, immunoprecipitation/input>2). (G-I) m⁶A-seq analysis of ALKBH5 knockdown NOMO1 cells. (G) The distribution of total m⁶A peaks in the indicated regions of mRNA transcripts in the control and ALKBH5-knockdown cells. (H) The distribution of differential m⁶A peaks (i.e., those with significant changes upon ALKBH5 manipulation). 5’UTR (150 nt) represents the first 150 nt of 5’ end of 5’UTR, while 5’UTR (Rest) represents the remaining regions of 5’UTR. (I) GSEA of the genes with significantly increased m⁶A abundance in ALKBH5 knockdown cells (p<0.05). (J) Integrative analysis to identify transcriptome-wide potential targets of ALKBH5 in AML. Left: potential positive targets of ALKBH5. Right: potential negative targets of ALKBH5. KD-Down and KD-Up: genes with significantly decreased and increased expression, respectively, upon ALKBH5 knockdown in both NOMO1 and MOLM13 cells as detected by RNA-seq (RPKM>1, fold change >1.5). RIP-seq: genes with significant enrichment in RIP samples (RPKM>1, immunoprecipitation/input>2). KD-m⁶A-Hyper: genes with significantly higher m⁶A abundance in ALKBH5 knockdown cells (p<0.05). (K) Expression change validation of potential positive and negative targets of ALKBH5 by qPCR. (L) ALKBH5-RIP qPCR validation of ALKBH5 binding of representative positive and negative targets. (M) Gene-specific m⁶A-RIP qPCR validation of m⁶A level changes of representative positive targets and negative targets. Mean±SD values are shown for Figures 5C, K, L and M.*p < 0.05; **p <0.01; ***p < 0.001; t test. See also Figure S5.

Figure 6.. ALKBH5 regulates TACC3 expression via affecting its mRNA stability.

(A) Kaplan-Meier survival analysis of TACC3 in the TCGA AML dataset. The p value was detected by the log-rank test. (B) The RNA (top) and m⁶A (bottom) abundance in TACC3 mRNA transcripts in ALKBH5 knockdown and control AML cells as detected by RNA-seq and m⁶A-seq. (C-E) Western blots of ALKBH5 and TACC3 in ALKBH5 stable knockdown MMC6 cells (C), ALKBH5 inducible knockdown NOMO1 cells (Dox induction for 4 days) (D) and ALKBH5 stable knockdown primary AML cells (E). VINCULIN or GAPDH was used as a loading control. (F) Western blots of ALKBH5 and TACC3 in MMC6 cells transduced with lentiviruses expressing empty vector (Vector) or wild-type ALKBH5 protein (A5-WT) or m⁶A demethylase-inactive mutant (A5-Mut). GAPDH was used as a loading control. (G-H) qPCR detection of Tacc3 expression in Alkbh5 WT or Homo KO mouse BM cells (G) and in primary mouse MA9 AML cells with shNS or Alkbh5 shRNA (shA5-#b) (H). (I-K) mRNA stability profiling. (I) Cumulative distribution of global transcript stability changes in shNS or shA5-#1 transduced NOMO1 cells. (J) Distribution of genes with significant half-life change in ALKBH5 knockdown cells compared to control cells. (K) Pathway analysis by GSEA showing the major pathways in which the genes with significantly shortened half-lives upon ALKBH5 knockdown are enriched. (L-N) The mRNA half-life (t_1/2) of TACC3 in MOLM13 cells (L) and NOMO1 cells (M) transduced with shNS or ALKBH5 shRNA (shA5-#1), and in NOMO1 cells transduced with empty vector (EV) or wild-type ALKBH5 (A5-WT) or ALKBH5 mutant (A5-Mut) (N). (O-Q) Western blots of ALKBH5, TACC3, MYC and P21 in AML cells transduced with shNS or shALKBH5 (shA5-#1) (O) or with inducible shNS (i-shNS) or shALKBH5 (i-shA5-#3) (P and Q). VINCULIN was used as a loading control. Mean±SD values are shown for Figures 6G–H and L–N. *p < 0.05; ***p<0.001, t test. See also Figure S6.

Figure 7.. TACC3 is a functionally important target of ALKBH5 in AML.

(A-B) Effects of ALHBK knockdown on cell growth/proliferation assays (A) and apoptosis (B) in AML cells. (C-D) Western blots of TACC3, MYC and P21 in NOMO1 cells (C) and MMC6 cells (D) expressing shNS or TACC3 shRNAs. VINCULIN was used as a loading control. (E) Relative levels of Tacc3 mRNA in mouse MA9 AML cells transduced with shNS or individual Tacc3 shRNAs (shTacc3-#a and shTacc3-#b). (F) Effects of Tacc3 knockdown on the viability/proliferation of mouse MA9 AML cells. (G) Effects of Tacc3 knockdown on the colony-forming/replating capacity of Mouse MA9 AML cells. Colony forming cell counts at each round of plating are shown (n=3). (H) In vitro limiting dilution assays (LDAs). Logarithmic plot showing the percentage of non-responding wells at different doses. Non-responding wells, wells not containing colony forming cells. The estimated LSC/LIC frequency is calculated by ELDA and shown on the right. The p value was detected by the log-rank test. (I-J) MMC6 cells were transduced with shNS or ALKBH5 shRNA (shA5), together with an empty (vector) or TACC3-encoding lentivirus as indicated. After drug selection, those co-transduced cells were seeded into 96-well plates for cell growth/proliferation assays (I). (J) Western blots of ALKBH5, TACC3, MYC and P21. VINCULIN was used as a loading control. (K) Proposed model demonstrating the role and underlying mechanism(s) of ALKBH5 in AML pathogenesis and LSC/LIC self-renewal. Mean±SD values are shown for Figures 7A, E–G and I. **p <0.01; ***p<0.001, t test. See also Figure S7.