-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

3925 Inhibiting the Mitochondrial RNA Degradosome Complex SUV3 and Pnpase Increases dsRNA in the Cytoplasm, Triggers a Viral Mimicry Response and Kills AML Cells and Progenitors

Program: Oral and Poster Abstracts
Session: 602. Myeloid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acute Myeloid Malignancies, AML, Translational Research, Diseases, Myeloid Malignancies
Monday, December 12, 2022, 6:00 PM-8:00 PM

Geethu Emily Thomas, PhD, MSc, BSc1, Kazem Nouri, PhD2*, Rose Hurren, BSc1*, Yongran Yan, MSc3*, Neil MacLean1*, Yulia Jitkova, PhD1*, Li Ma2*, Xiaoming Wang4*, Chaitra Sarathy, PhD1*, Andrea Arruda, MSc5*, Mark D. Minden, MD, PhD6, Vito Spadevecchio, B.A7* and Aaron D. Schimmer, MD, PhD6

1Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada
2Princess Margaret Cancer Research, Toronto, ON, Canada
3Princess Margaret Cancer Center, University Health Network, Toronto, ON, Canada
4Princess Margaret Cancer Research, University Health Network, Toronto, ON, Canada
5University Health Network, Toronto, ON, Canada
6Division of Medical Oncology and Hematology, University Health Network, Toronto, ON, Canada
7Interlinked Therapeutics LLC, Portland, OR

Eukaryotic cells have two separate genomes; nuclear DNA organized in chromosomes, and the circular mitochondrial DNA located within mitochondria. Mitochondrial DNA is comprised of a double-stranded circular genome that is 16.6 kB in length, lacks introns, and encodes two rRNAs, 22 t-RNAs and 13 of the 90 proteins in the mitochondrial respiratory chain. To maintain homeostasis, mitochondria possess RNA degradation machinery to regulate mitochondrial RNA turnover. The ATP-dependent helicase, SUV3 (encoded by the gene SUPV3L1), and the exonuclease PNPase (encoded by the gene PNPT1) function in complex to degrade mitochondrial dsRNA.

We identified PNPT1 and SUPV3L1 through an unbiased in silico screen using the ITX machine learning platform to identify novel targets in AML. Further simulations revealed inhibition of these genes could lead to significant enrichment of Response to exogenous dsRNA, Response to virus, and RNA catabolic process ontologies.

By immunoblotting PNPase and SUV3 protein were increased in 7/7 AML patient samples and 13/13 of AML cell lines compared to the normal hematopoietic cells. Analysis of the TARGET AML dataset revealed that AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall survival and event free survival respectively.

To study the importance of PNPT1 and SUPV3L1 in AML, we knocked down or knocked out these genes with shRNA or sgRNA in AML cells. Genetic knockdown or knock out of PNPT1 or SUPV3L1 decreased growth and viability of OCI-AML2, TEX, K562, U937, NB4 and OCI-AML-8227 leukemia cells. Moreover, SUPV3L1 & PNPT1 ranked top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (https://depmap.org/portal). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of OCI-AML2, TEX and U937 cells. Demonstrating the functional importance of PNPT1 & SUPV3L1 on leukemia initiating cells in vivo, genetic knockdown of PNPT1 and SUPV3L1 significantly reduced engraftment of TEX cells into the marrow of immune deficient mice. Finally, primary AML cells with SUPV3L1 knockdown had reduced engraftment into the marrow of immune deficient mice.

Mechanistically, knockdown of PNPT1 and SUPV3L1 in AML2 cells increased levels of cytoplasmic dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased cytoplasmic dsRNA in 143B cells, but not Rho(0) 143B cells that lack mitochondrial DNA. Increased cytoplasmic dsRNA can mimic viral infection and trigger a type 1 Interferon response. Knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) associated with a type 1 interferon response compared to control.

In summary, the RNA degradosome complex proteins SUPV3L1 and PNPT1 are overexpressed in AML and are essential for AML cells and stem/progenitors. These enzymes regulate the levels of mitochondrial dsRNA and their inhibition leads to increased cytoplasmic dsRNA and triggers a viral mimicry response.

Disclosures: Spadevecchio: Interlinked Therapeutics, LLC: Current Employment. Schimmer: Novartis: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Takeda Pharmaceuticals: Consultancy, Honoraria, Research Funding; Medivir AB: Research Funding; BMS: Consultancy, Honoraria, Research Funding; Otsuka Pharmaceuticals: Consultancy, Honoraria; UHN: Patents & Royalties: the use of DNT cells to treat AML.

*signifies non-member of ASH