The development and regulation of malignant self-renewal remains an unresolved issue. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2'-O-methylation regulate leukemia stem cell (LSC) activity in vivo. A comprehensive analysis of the rRNA 2'-O-methylation landscape of 94 acute myeloid leukemia (AML) patients revealed dynamic 2'-O-methylation specifically at exterior sites of ribosomes. rRNA 2'-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of 2'-O-methyltransferase FBL induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2’-O-methylation redirected the ribosome translation program towards amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2'-O-Me at specific sites on ribosomal RNAs shifts translational preferences and controls AML-LSC self-renewal.

<div>Abstract<p>The development and regulation of malignant self-renewal remain unresolved issues. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2′-O-methylation regulate leukemia stem cell (LSC) activity <i>in vivo</i>. A comprehensive analysis of the rRNA 2′-O-methylation landscape of 94 patients with acute myeloid leukemia (AML) revealed dynamic 2′-O-methylation specifically at exterior sites of ribosomes. The rRNA 2′-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of the 2′-O-methyltransferase fibrillarin (FBL) induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2′-O-methylation redirected the ribosome translation program toward amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2′-O-methylation at specific sites on rRNAs shifts translational preferences and controls AML LSC self-renewal.</p>Significance:<p>We establish the complete rRNA 2′-O-methylation landscape in human AML. Plasticity of rRNA 2′-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function.</p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-13-2-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 247</a></i></p></div>

<div>Abstract<p>The development and regulation of malignant self-renewal remain unresolved issues. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2′-O-methylation regulate leukemia stem cell (LSC) activity <i>in vivo</i>. A comprehensive analysis of the rRNA 2′-O-methylation landscape of 94 patients with acute myeloid leukemia (AML) revealed dynamic 2′-O-methylation specifically at exterior sites of ribosomes. The rRNA 2′-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of the 2′-O-methyltransferase fibrillarin (FBL) induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2′-O-methylation redirected the ribosome translation program toward amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2′-O-methylation at specific sites on rRNAs shifts translational preferences and controls AML LSC self-renewal.</p>Significance:<p>We establish the complete rRNA 2′-O-methylation landscape in human AML. Plasticity of rRNA 2′-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function.</p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-13-2-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 247</a></i></p></div>

<p>Supplementary Figure S1 shows enrichment of FBL in LSC, correlation of FBL with LSC signature and rRNA 2'-O-Me in healthy hematopoietic cells. Supplementary Figure S2 shows the association of rRNA 2’-O-Me with LSC and hematopoietic differentiation signatures. Supplementary Figure S3 shows strategy for LSC sorting and distribution of LSC methylation sites on ribosomes. Supplementary Figure S4 shows the effect of FBL knockdown on rRNA 2’-O-methylation and in vitro proliferation of leukemia cells. Supplementary Figure S5 show the effect of FBL overexpression on in vivo engraftment of primary AML cells. Supplementary Figure S6 shows the effect of FBL knockdown on nascent proteome and metabolism of AML cells. Supplementary Figure S7 shows the ribosome footprinting analysis after FBL knockdown. Supplementary Figure S8 shows the association of Gm1447 with LSC signature and the effect of Gm1447 supression on cellular amino acid levels. Supplementary Figure S9 shows regulatory effect of Gm1447 on in vivo leukemic self-renewal.</p>

<p>Supplementary Figure S1 shows enrichment of FBL in LSC, correlation of FBL with LSC signature and rRNA 2'-O-Me in healthy hematopoietic cells. Supplementary Figure S2 shows the association of rRNA 2’-O-Me with LSC and hematopoietic differentiation signatures. Supplementary Figure S3 shows strategy for LSC sorting and distribution of LSC methylation sites on ribosomes. Supplementary Figure S4 shows the effect of FBL knockdown on rRNA 2’-O-methylation and in vitro proliferation of leukemia cells. Supplementary Figure S5 show the effect of FBL overexpression on in vivo engraftment of primary AML cells. Supplementary Figure S6 shows the effect of FBL knockdown on nascent proteome and metabolism of AML cells. Supplementary Figure S7 shows the ribosome footprinting analysis after FBL knockdown. Supplementary Figure S8 shows the association of Gm1447 with LSC signature and the effect of Gm1447 supression on cellular amino acid levels. Supplementary Figure S9 shows regulatory effect of Gm1447 on in vivo leukemic self-renewal.</p>