Mitochondrial RNA pseudouridine reprograms metabolism and induces immunosuppression in myeloid leukemia Free

Pseudouridine (Ψ) is the most prevalent RNA modification in human cells. Although Ψ was among the first post-transcriptional modifications discovered (1951), its biological functions remain poorly understood. More recently, Ψ was incorporated into COVID-19 mRNA vaccine, where it enhances RNA stability and reduces innate immune sensing. However, the precise roles of RNA Ψ in cancer, including leukemia, are largely unknown. Here, we identify RNA Pseudouridine Synthase D4 (RPUSD4) as a novel vulnerability in acute myeloid leukemia (AML) via directly catalyzing mitochondrial RNA (mt-RNA) Ψ formation. We further provide proof-of-concept evidence that targeting RPUSD4 suppresses leukemogenesis and stimulates anti-leukemia immunity.

To investigate whether RNA Ψ dysregulation is involved in leukemia, we first compared RNA Ψ levels between primitive CD34⁺ cells (LSC-enriched) and CD34^- bulk cells from multiple primary AML specimens. CD34⁺ AML cells exhibited significantly elevated RNA Ψ levels. We also examined Ψ levels across various RNA types in AML cells, identifying that mt-RNA contained much higher Ψ levels than other RNA types. These findings imply that RNA Ψ, particularly mt-RNA Ψ, may play a role in LSC biology and leukemogenesis.

Further unbiased CRISPR screening revealed RPUSD4 as the most critical Ψ synthase for LSC maintenance. Large-scale transcriptomic and patient data analyses showed that RPUSD4 is highly expressed in AML specimens (P = 3.5 × 10^-9), particularly within LSCs (P = 1.4 × 10^-18). RPUSD4 knockout (KO) significantly suppressed AML proliferation, reduced mitochondrial OxPhos, and decreased LSC frequency in vitro and substantially delayed AML progression in vivo (MLL-r and FLT3^ITD models). Of note, RPUSD4 KO-mediated anti-leukemia effects could be fully rescued by wild-type but not catalytically inactive RPUSD4 both in vitro and in vivo, demonstrating that Ψ synthase activity is essential for its function in leukemia.

To profile RPUSD4-dependent Ψ sites, we applied two orthogonal technologies, direct nanopore sequencing and PRAISE sequencing (PMID: 36997645), both capable of detecting Ψ at single-base resolution. These approaches converged on a single RPUSD4-dependent Ψ at position 3,067 on mt-16S rRNA. This Ψ site is essential for maintaining rRNA stability. RPUSD4 KO induced mt-16S rRNA decay, thereby suppressing mitochondrial translation. This led to OxPhos inhibition, growth suppression, and LSC eradication in AML.

Mechanistically, RPUSD4 is exclusively localized in mitochondria, where it directly interacts with the mt-RNA exonuclease PNPase. Under physiological conditions, PNPase resides within mitochondria and degrades mt-RNA to prevent accumulation of mitochondrial double-stranded RNA (mt-dsRNA). Upon RPUSD4 depletion, PNPase translocates from mitochondria to the cytosol, resulting in two major consequences:

(i) mRNA decay:Cytosolic PNPase degrades mRNAs, particularly the nuclear-encoded mitochondrial mRNAs, such as DHODH(Dihydroorotate dehydrogenase). By retrogradely upregulating DHODH, RPUSD4 promotes de novo pyrimidine synthesis in AML.

(ii) Aberrant mt-dsRNA accumulation: The buildup and cytoplasmic release of mt-dsRNA activates “viral mimicry” response that sensitizes AML cells to T cell cytotoxicity.

Finally, to therapeutically target RPUSD4, we developed an RNA-based inhibitor, CpG-siRNA^RPUSD4, by conjugating a specific RPUSD4 siRNA to a Toll-like receptor (TLR9) ligand. CpG-siRNA^RPUSD4 is specifically internalized by TLR9⁺ cells, including AML cells, thereby maximizing selectivity and minimizing toxicity. Preclinical studies demonstrate that pharmacologically targeting RPUSD4 significantly prolonged survival across multiple AML models, including patient-derived xenograft (PDX) models, and rendered AML cells more susceptible to T cell–mediated cytotoxicity. Furthermore, the RPUSD4 inhibitor exhibited a promising synergistic effect with DHODH inhibitor in treating relapsed AML in vivo. For instance, in a representative relapsed PDX model, median survival times (in days) were: Ctrl – 28, RPUSD4 inhibitor – 36, DHODH inhibitor – 39, and combination – over 60.

In conclusion, our findings uncover a previously unrecognized role of RPUSD4 and mt-RNA Ψ in leukemic metabolism, anti-leukemia immunity, and LSC maintenance. This study also bridges a critical knowledge gap regarding the role of RNA Ψ in leukemogenesis.