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625 Mitochondrial RNA Methylation By METTL17 Rewires Metabolism and Regulates Retrograde Mitochondrial-Nuclear Communication in AML

Program: Oral and Poster Abstracts
Type: Oral
Session: 602. Myeloid Oncogenesis: Basic: Models and Mechanisms in AML Biology
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acute Myeloid Malignancies, AML, Diseases, Myeloid Malignancies
Sunday, December 8, 2024: 4:30 PM

Xueer Wang, PhD1, Honghai Zhang2*, Lei Dong3*, Xiaoxu Zhang2*, Meilin Xue4*, Lili Ren2*, Hongjie Bi2*, Lucy Y. Ghoda5*, Mark Wunderlich, MS6*, James C. Mulloy, PhD7*, Chun-Wei David Chen, PhD8, Ling Li, PhD9, Lin Xu3*, Bin Zhang5, Guido Marcucci10, Steven T. Rosen, MD11, Nora Heisterkamp12*, Marcin Kortylewski, PhD13*, Jianjun Chen, PhD14 and Rui Su, PhD15*

1City of Hope National Medical Center, Duarte, CA
2City of hope national medical center, Monrovia, CA
3UT Southwestern Medical Center, Dallas, TX
4Ruijin Hospital, Shanghai, China
5Department of Hematological Malignancies Translational Science, Gehr Family Center for Leukemia Research, City of Hope Medical Center and Beckman Research Institute, Duarte, CA
6Division of Experimental Hematology and Cancer Biology, Cancer & Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
7Cincinnati Children's Hospital, Cincinnati, OH
8Department of Systems Biology, Beckman Research Institute, City of Hope, Duarte, CA
9Department of Hematological Malignancies Translational Science, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA
10City of Hope, Duarte, CA
11Department of Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, CA
12City of Hope Comprehensive Cancer Center, Monrovia, CA
13Department of Immuno-Oncology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA
14Gehr Family Center for Leukemia Research, City of Hope, Duarte, CA
15City of hope, Monrovia, CA

Leukemia stem cells (LSCs) are recognized as the root cause of AML pathogenesis. LSCs heavily depend on mitochondrial oxidative phosphorylation (OxPhos) for energy production. Recent studies, including our own, suggest that RNA modifications are critical for post-transcriptional gene regulation during leukemogenesis, and targeting RNA modifications is a promising strategy to eradicate LSCs. Intriguingly, mitochondria transcribe mitochondrial RNAs (mt-RNAs) also decorated with various modifications from their own circular genome. If such mt-RNA modifications contribute to AML was unclear. Here we show that methyltransferase like 17 (METTL17), an enzyme specifically catalyzing mt-RNA cytosine methylation, represents a novel vulnerability that can be targeted to eliminate LSCs and AML.

To investigate proteins responsible for mt-RNA modification, we initially surveyed overlap between human RNA-binding proteins (RBPs; 1,515) and mitochondrial proteins (1,158), identifying 183 mitochondrial RBPs (mt-RBPs). Notably, among these, METTL17 stood out as most relevant for AML because (i) expression is significantly elevated in AML patients compared to healthy controls (P = 8.43*10-8); (ii) this associates with poor survival in AML (TCGA; P = 0.0085; Hazard ratio = 2.1); (iii) it is highly expressed in CD34+ LSCs in contrast to bulk AML cells; and (iv) it is essential for AML survival as determined by CRISPR screening. We then utilized CRISPR/Cas9 to knock out (KO) endogenous METTL17 expression in AML cells including PDX cells, with different driver mutations (MLL-r, FLT3ITD, and DNMT3A mutation), for both in vitro and in vivo studies. We also overexpressed METTL17 wildtype (WT), loss-of-function (LoF) mutant, and depletion of mitochondrial targeting sequence (ΔMTS) mutant in METTL17 KO cells to determine to which extent its functions rely on its enzymatic activity and mitochondrial localization. METTL17 KO significantly inhibited AML cell proliferation, induced cell cycle arrest, reduced OxPhos, and decreased LSC frequency in vitro, and notably suppressed leukemogenesis in vivo. Moreover, those effects were fully reversed by WT METTL17, but not by LoF or ΔMTS mutants, demonstrating that both mitochondrial localization and methyltransferase activity are responsible for its contributions to AML pathogenesis.

To elucidate the underlying mechanisms, we conducted multiple-omics analyses. CLIP-seq demonstrated a robust interaction between METTL17 and mt-RNAs, particularly 12S rRNA. By characterizing methylation sites at base resolution, we determined that METTL17 directly deposits cytosine N4-methylation (m4C) at position 839 (m4C839), and N5-methylation (m5C) at position 841 (m5C841) on mt-12S rRNA. Such methylation is needed for mt-12S rRNA stability: METTL17 KO caused decay of 12S rRNA. Its KO reduced mitochondrial protein synthesis and OxPhos, thereby inhibiting AML proliferation and eradicating LSCs. Metabolomics analysis revealed that METTL17 KO suppressed the tricarboxylic acid cycle and reduced acetyl-CoA levels, leading to decreased nuclear histone H3 lysine 27 acetylation (H3K27ac), which in turn influenced the expression of key nuclear genes. Integrative analysis of RNA-seq, H3K27ac CUT&RUN-seq, and H3K27ac super-enhancers, along with experimental validation demonstrated CCND3 as a functionally essential target of the METTL17/H3K27ac axis.

Finally, to apply our fundamental discoveries to potential clinical applications, we developed a specific and potent inhibitor by linking METTL17 siRNA to a Toll-like receptor (TLR9) ligand, CpG-D19 oligodeoxynucleotide (CpG-siRNAMETTL17). Our prior studies demonstrated that CpG-siRNAs are exclusively internalized by the restricted population of immune cells that are TLR9+, including AML cells. Proof-of-concept studies showed that pharmacologically targeting METTL17 significantly inhibited AML growth, reduced OxPhos, and eradicated LSCs in vitro, and substantially prolonged survival of AML models in vivo. Importantly, CpG-siRNAMETTL17 had no effects on either growth or metabolism of normal CD34+ HSCs, indicating minimal toxicity.

In conclusion, our findings reveal METTL17 as a promising treatment target that makes use of the specific metabolic vulnerability of AML LSCs. We also introduce the novel concept that mt-RNA modification regulates retrograde mitochondrial-nuclear communication.

Disclosures: Rosen: Astra Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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