AML Cells Have Increased Mitochondrial RNA Degradation and Inhibiting This Degradation Promotes Cell Differentiation, Decreases Viability, and Increases Sensitivity to Immune-Mediated Killing
Authors: Thomas Geethu Emily , Nouri Kazem , Hurren Rose , Lee JongBok , Voisin Veronique , Yan Yongran , Maclean Neil , Gronda Marcela , Jitkova Yulia , Barbieri Taylor , Ma Li , Wang Xiaoming , Arruda Andrea , Minden Mark D. , Zhang Li , Schimmer Aaron D 
Published: December 2024

Eukaryotic cells have two separate genomes; nuclear DNA organized in chromosomes and circular mitochondrial DNA within mitochondria. Mitochondrial DNA encodes 13 proteins, which are all subunits of the respiratory chain and are necessary for oxidative phosphorylation and mitochondrial energy metabolism. We identified increased expression of mitochondrial encoded genes by RT-PCR in primary AML cells (n=14) compared to normal hematopoietic bulk and progenitor cells (n=7), consistent with AML's increased reliance on oxidative phosphorylation. We also discovered that degradation and turnover of mitochondrial mRNA were increased in primary AML cells and AML cell lines.

Degradation of mitochondrial RNA is mediated by an ATP-dependent helicase, SUPV3L1, and an exonuclease, PNPT1. SUPV3L1 and PNPT1 mRNA and protein were increased in primary AML cells compared to normal hematopoietic cells. Knocking down SUPV3L1 and PNPT1 in AML prevented mitochondrial RNA degradation, as evidenced by increased levels of polyA tagged mitochondrial RNA.

The transcription and turnover of mitochondrial genes generates double stranded RNA (dsRNA) sequences. Knockdown of SUPV3L1 and PNPT1 increased dsRNA in primary AML patient samples and AML cell lines. We immunoprecipitated and sequenced the dsRNA after SUPV3L1 and PNPT1 knockdown. The dsRNA after knockdown of SUPV3L1 and PNPT1 was enriched (275-fold) for mitochondrial over nuclear sequences, demonstrating a mitochondrial source for the dsRNA.

We also examined changes in gene expression after knockdown of PNPT1 and SUPV3L1. Knockdown of SUPV3L1 and PNPT1 upregulated genes associated with an interferon response and viral infection, consistent with a type 1 interferon response and viral mimicry.

Induction of viral mimicry can induce differentiation and reduce viability of AML cells. We demonstrated that the knockdown of SUPV3L1 and PNPT1 in AML cells increased non-esterase staining, a marker of differentiation. We also observed that genetic knockdown or knockout of SUPV3L1 and PNPT1 decreased the growth and viability of OCI-AML2, TEX, K562, U937, NB4 and 8227 cell lines and clonogenic growth of AML cells OCI-AML2, TEX and U937. Knockdown of SUPV3L1 and PNPT1 also reduced engraftment of TEX cells into the marrow of immune-deficient mice. Finally, knocking down of SUPV3L1 in primary AML cells reduced their engraftment into the marrow of immune-deficient mice. In contrast, knockdown of SUPV3L did not alter the primary engraftment of CD34+ enriched cord blood cells.

Finally, we explored sensitivity of AML cells to immune-mediated killing as induction of viral mimicry pathways can enhance sensitivity to T-cell mediated cytotoxicity. Knockdown of of SUPV3L1 and PNPT1 increased T-cell mediated killing of AML cells in vitro. In addition, using immunocompetent and T-cell depleted Balb/c mouse models engrafted with murine A20 cells, we demonstrated that endogenous T cells were required to achieve the greatest reduction in leukemia after SUPV3L1 knockdown.

Thus, in summary, AML cells have increase mitochondrial RNA degradation mediated by PNPT1 and SUPV3L1. Genetic depletion of PNPT1 and SUPV3L1 increases dsRNA leading to an interferon response, AML differentiation, and increased sensitivity to T-cell mediated killing.