KMT2D Deficiency Promotes Myeloid Leukemias which Is Vulnerable to Ribosome Biogenesis Inhibition

Abstract

KMT2C and KMT2D are the most frequently mutated epigenetic genes in human cancers. While KMT2C is identified as a tumor suppressor in acute myeloid leukemia (AML), the role of KMT2D remains unclear in this disease, though its loss promotes B cell lymphoma and various solid cancers. Here, it is reported that KMT2D is downregulated or mutated in AML and its deficiency, through shRNA knockdown or CRISPR/Cas9 editing, accelerates leukemogenesis in mice. Hematopoietic stem and progenitor cells and AML cells with Kmt2d loss have significantly enhanced ribosome biogenesis and consistently, enlarged nucleolus, increased rRNA and protein synthesis rates. Mechanistically, it is found that KMT2D deficiency leads to the activation of the mTOR pathway in both mouse and human AML cells. Kmt2d directly regulates the expression of Ddit4, a negative regulator of the mTOR pathway. Consistent with the abnormal ribosome biogenesis, it is shown that CX-5461, an inhibitor of RNA polymerase I, significantly restrains the growth of AML with Kmt2d loss in vivo and extends the survival of leukemic mice. These studies validate KMT2D as a de facto tumor suppressor in AML and reveal an unprecedented vulnerability to ribosome biogenesis inhibition.

Keywords: KMT2D; acute myeloid leukemia; epigenetics; mTOR; ribosome biogenesis.

Figure 1

Kmt2d deficiency by shRNAs promotes AML in mice. A) Expression levels of KMT2D in AML and normal samples. Left, data were analyzed from RNA‐seq data (GSE48173) with 17 CD34⁺ cord blood and 43 AML samples; Right, data were analyzed from microarray data (GSE1159) with eight healthy donors (five normal bone marrow and three CD34⁺ cell samples) and 285 AML samples; *p < 0.05, ***p < 0.001 (two‐tailed Wilcoxon rank‐sum test). B) Survival curves of AML patients stratified by high and low expression of KMT2D in the TCGA‐LAML (left) and Beat AML (right) cohort, respectively. The cut‐off values were determined by maximally selected rank statistics. p Values were determined by the log‐rank test. C) Schematic experimental design for mouse modeling using shRNA technique. Trp53 ^−/− mouse HSPCs were transduced with GFP‐linked shKmt2d/shRen and mCherry‐linked shNf1, and then transplanted into sub‐lethally irradiated syngeneic mice. D) Kaplan–Meier survival curves of mice transplanted with Trp53 ^−/− HSPCs transduced with shNf1 and shRen (blue; n = 10), shKmt2d_#1 (red; n = 10), or shKmt2d_#2 (orange; n = 10). **p < 0.01, ***p < 0.001 (log‐rank test). The results were the combination of two independent trials. E) White blood cell (WBC), hemoglobin (Hb), and platelet (PLT) counts of shKmt2d and shRen mice 7 weeks post‐transplant. F) Representative flow cytometric profiles showing the expression of fluorescent markers (GFP and mCherry), myeloid lineage markers (CD11b and Gr‐1), lymphoid lineage markers (B220 and CD3ε), and stem cell marker (c‐Kit) in bone marrow cells of sacrificed TNK (Trp53 ^−/−; shNf1; shKmt2d) mice. G) Representative images of histological analyses of blood, spleen, liver, and bone marrow of sacrificed TNK mice. H) Kaplan–Meier survival curves of secondary transplants from two independent primary leukemia cells of each TNK mouse (n = 4 per group). I) Relative mRNA levels of Kmt2d in bone marrow cells of sacrificed control TN (Trp53 ^−/−; shNf1; shRen) and TNK mice were quantified by qRT‐PCR (normalized to Actin; n = 3 per group). J) Western blotting analyses showing the H3K4me1 and H3K4me2 levels in bone marrow cell lysates from sacrificed TN and TNK mice. E,I) Graph represents the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant (unpaired two‐tailed t‐test).

Figure 2

Kmt2d mutation by CRISPR/Cas9 promotes AML in mice. A) Schematic experimental design for mouse modeling using CRISPR/Cas9 system. Trp53 ^−/−; Cas9 mouse HSPCs were transduced with mCherry‐linked sgKmt2d‐sgNf1‐sgCas9 or sgScramble‐sgNf1‐sgCas9, and then transplanted into sub‐lethally irradiated syngeneic mice. B) Kaplan–Meier survival curves of mice transplanted with Trp53 ^−/−; Cas9 HSPCs transduced with sgScramble‐sgNf1‐sgCas9 (blue; n = 5), sgKmt2d_#1‐sgNf1‐sgCas9 (red, n = 5), or sgKmt2d_#2‐sgNf1‐sgCas9 (orange; n = 5). **p < 0.01 (log‐rank test). C) WBC, Hb, and PLT counts of sgScramble and sgKmt2d mice 2 months post‐transplant. Graph represents the mean ± SD; **p < 0.01, ***p < 0.001, ns, not significant (unpaired two‐tailed t‐test). D) Representative flow cytometric profiles showing the expression of CD11b/Gr‐1, B220/CD3ε and c‐Kit in bone marrow cells of sacrificed TNKC (Trp53 ^−/−; sgNf1; sgKmt2d; sgCas9) mice. E) Representative images of histological analyses of blood, spleen, liver, and bone marrow of sacrificed TNKC mice. F) T7 endonuclease I assay on Kmt2d in bone marrow cells of sacrificed TNKC mice. G) Mutation analyses of the Kmt2d regions targeted by CRISPR/Cas9 of TNKC bone marrow cells. Representative Sanger sequences of single TA clones.

Figure 3

Kmt2d negatively regulates ribosome biogenesis in AML. A) Relative mRNA levels of Kmt2d in AML cells obtained from TRE‐rtTA‐driven inducible Kmt2d knockdown mice treated with doxycycline (shKmt2d, KD) or without doxycycline (Kmt2d‐restored, RS) were quantified by qRT‐PCR (normalized to Hprt; n = 3 per group). B) The effect of Kmt2d knockdown on cell growth (n = 3 per group). C) The effect of Kmt2d knockdown on cell morphology. Representative pictures performed on Liu's‐stained cytospins (left) and quantitation of cell size and nuclear size (right) in Kmt2d restored and knockdown AML cells (n = 100 per group). D) The effect of Kmt2d knockdown on nucleolus size. Representative transmission electron microscopy images (left) and quantitation of nucleolus size (right) in Kmt2d restored and knockdown AML cells (n = 20 per group). E) The effect of Kmt2d knockdown on the intensity of nucleolar protein fibrillarin (red). Representative immunofluorescence images (left) and quantitation of fluorescence intensity per nucleus (right) in Kmt2d restored and knockdown AML cells (n = 100 per group). F) The effect of Kmt2d knockdown on rRNA synthesis. Cells were labeled with 5‐FUrd for 30 min and immunostained with an antibody against BrdU. Representative immunofluorescence images (left) and quantitation of fluorescence intensity per nucleus (right) in Kmt2d restored and knockdown AML cells (n = 100 per group). G) The effect of Kmt2d knockdown on relative levels of 18S and 28S rRNA was quantified by qRT‐PCR in Kmt2d restored and knockdown AML cells (normalized to Actin; n = 3 per group). H) The effect of Kmt2d knockdown on protein synthesis was performed by OPP incorporation assay in Kmt2d restored and knockdown AML cells (n = 3 per group). Representative flow cytometric profile (left) and quantitation of OPP MFI (right) (n = 3 per group). I) GO analysis of significantly upregulated genes in Kmt2d knockdown AML cells compared to restored cells (log2‐fold change > 0.5, p < 0.05). p adjust values were determined by Benjamini–Hochberg correction (p adjust < 0.05). J) GSEA showing the positive enrichment of the KEGG_RIBOSOME, GO_NUCLEOLAR_PART, GO_RRNA_METABOLIC_PROCESS, and REACTOME_TRANSLATION gene sets in Kmt2d knockdown AML cells compared to restored cells. C–F) Quantitation was analyzed by Image J. B–H) Graph represents the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired two‐tailed t‐test).

Figure 4

Kmt2d regulates ribosome biogenesis through the mTOR signaling pathway. A) GSEA showing the positive enrichment of the HALLMARK_MTORC1_SIGNALING gene set in Kmt2d knockdown AML cells compared to restored cells. B) Representative western blotting showing the phosphorylation levels of ribosomal protein S6 in Kmt2d restored and knockdown AML cells. Leukemia cells obtained from two inducible shKmt2d‐driven AML mice were tested. C) The effect of mTOR inhibitor rapamycin on the intensity of nucleolar protein fibrillarin (red). Representative immunofluorescence images (left) and quantitation of fluorescence intensity per nucleus (right) in rapamycin‐treated and vehicle‐treated Kmt2d knockdown AML cells (n = 50 per group). D) The effect of rapamycin on cell morphology. Representative pictures performed on Liu's‐stained cytospins (left) and quantitation of cell size and nuclear size (right) of rapamycin‐treated and vehicle‐treated Kmt2d knockdown AML cells (n = 100 per group). E) Representative Integrative Genomics Viewer browser tracks of KMT2D, H3K4me1, and H3K4me2 peaks on Ddit4 locus and Ddit4 mRNA reads in Kmt2d restored and knockdown AML cells. F) Relative mRNA levels of Ddit4 in Kmt2d restored and knockdown AML cells were quantified by qRT‐PCR (normalized to Hprt, n = 3 per group). G) Representative western blotting showing the effect of Ddit4 overexpression on phosphorylation levels of ribosomal protein S6 in Kmt2d knockdown AML cells. Leukemia cells obtained from two inducible shKmt2d‐driven AML mice were tested. H) The effect of Ddit4 overexpression on the intensity of nucleolar protein fibrillarin (red) in Kmt2d knockdown AML cells. Representative immunofluorescence images (left) and quantitation of fluorescence intensity per nucleus (right) (n = 90 per group). I) The effect of Ddit4 overexpression on total RNA from Kmt2d knockdown AML cells was separated by nondenaturing agarose gel (1%) electrophoresis. The positions of 18S and 28S ribosomal RNA are indicated. J) The effect of Ddit4 overexpression on protein synthesis was performed by OPP incorporation assay in Kmt2d knockdown AML cells. Representative flow cytometric profile (left) and quantitation of OPP MFI (right) (n = 3 per group). K) The effect of Ddit4 overexpression on Kmt2d knockdown AML cell growth (n = 3 per group). B,C,D,H,I) Quantitation was analyzed by ImageJ. C,D,F,H,J,K) Graph represents the mean ± SD, **p < 0.01, ***p < 0.001 (unpaired two‐tailed t‐test).

Figure 5

Kmt2d‐deficient AML is sensitive to the ribosome biogenesis inhibitor. A) Schematic experimental design. TNK (Trp53 ^−/−; shNf1; shKmt2d) AML cells were transplanted into sub‐lethally irradiated recipient mice. CX‐5461 or vehicle (0.5% CMC‐Na) was administered orally (40 mg kg⁻¹) to recipient mice 10 days after transplant. The time points of drug administration are indicated. Mice were followed for AML development or sacrificed to analyze 4 weeks after transplant. B) Kaplan–Meier survival curves of leukemia mice treated with vehicle (blue; n = 7) or CX‐5461 (red; n = 7). ***p < 0.001 (log‐rank test). The results were the combination of two independent trials. C) WBC counts in the peripheral blood of CX‐5461 and vehicle‐treated leukemia mice 4 weeks after transplant (n = 6 per group). D) The percentage of GFP and mCherry double‐positive population in the peripheral blood of CX‐5461 and vehicle‐treated leukemia mice 4 weeks after transplant (n = 6 per group). E) Representative blood smears and bone marrow cytospins of CX‐5461 and vehicle‐treated leukemia mice 4 weeks after transplant. F) Representative images of spleens and livers (left) and quantitation of their weights (right) in CX‐5461 and vehicle‐treated leukemia mice 4 weeks after transplant (n = 6 per group). G) Representative images of histological analyses of bone marrow, spleen, and liver of CX‐5461 and vehicle‐treated leukemia mice 4 weeks after transplant. C,D,F) Graph represents the mean ± SD, ***p < 0.001 (unpaired two‐tailed t‐test).

Figure 6

KMT2D regulates ribosome biogenesis in human AML. A) GSEA showing the positive enrichment of the GO_RIBOSOME_BIOGENESIS, GO_RIBOSOME, GO_NUCLEOLAR_PART, and HALLMARK_MTORC1_SIGNALING gene sets in KMT2D low expression AML patients (n = 20) compared to KMT2D high expression ones (n = 122) in the TCGA‐LAML cohort. B) Scatter plot showing the negative correlation between expression levels of KMT2D and ribosome biogenesis‐related genes in the TCGA‐LAML cohort. p Values were determined by the two‐tailed Student's t‐test. r, Pearson's correlation coefficient. C) Representative pictures performed on Liu's‐stained cytospins (left) and quantitation of cell size and nuclear size (right) of sgScramble and sgKMT2D MOLM‐13 cell lines (n = 100 per group). D) Representative immunofluorescence images for nucleolar protein Fibrillarin (left) and quantitation of fluorescence intensity per nucleus (right) of sgScramble and sgKMT2D MOLM‐13 cell lines (n = 35 per group). E) Representative flow cytometric profile (left) and quantitation of OPP MFI in sgScramble and sgKMT2D MOLM‐13 cell lines (n = 3 per group). F) Western blotting showing the phosphorylation levels of ribosomal protein S6 in sgScramble and sgKMT2D MOLM‐13 cell lines. G) Schematic diagram showing the working model of KMT2D deficiency in leukemia cell growth. C,D,F) Quantitation was analyzed by ImageJ. C,D,E) Graph represents the mean ± SD, ***p < 0.001 (unpaired two‐tailed t‐test).