Oxidized Mitochondrial DNA Is a Catalyst and Biomarker of Pyroptotic Cell Death in Myelodysplastic Syndromes

Constitutive innate immune activation is a pathogenetic driver of Myelodysplastic Syndromes (MDS) that directs ineffective hematopoiesis by NLRP3 inflammasome (IFM) assembly and pyroptotic cell death. IFM activation involves recruitment of caspase-1 (casp1) through the adapter protein, ASC, to facilitate autocatalytic cleavage of the zymogen to its active form that is responsible for interleukin (IL)-1β maturation, membrane pore formation and pyroptosis. Oxidized mitochondrial DNA (ox-mtDNA) has been proposed to serve as an alarmin that can activate the IFM by interaction directly with NLRP3 or engagement by DNA sensors, Toll-like receptor 9 (TLR9) and Cyclic GMP-AMP synthase (cGAS). Upon cytolysis, ox-mtDNA is released, permitting interaction with pattern recognition receptors on neighboring cells (Grishman, Pediatric Research, 2012, Shimada, 2012, Immunity. Vollmer, 2004, Immunology). Here, we investigate ox-mtDNA as an IFM-activator and pyroptotic biomarker in MDS. Incubation of TLR9 expressing cell lines, SKM1 (high expresser) and U937 (moderate expresser) with 50ng/mL ox-mtDNA (ND1 gene, amplified with oxidized guanosine) induced IFM activation evidenced by increased p-NFkβ, casp1 and IL-1β cleavage, ASC oligomerization and liberation of ASC specks. Direct interaction of ox-mtDNA with NLRP3 was confirmed by NLRP3 immunoprecipitation followed by probing for mtDNA using ND1 and CYTB specific primers and GAPDH primers as negative genomic control; mtDNA oxidation status was confirmed by dot blot. Furthermore, significantly increased expression of interferon stimulated genes (ISG) was seen in MDS bone marrow (BM) specimens (p≤0.01) compared to normal donors indicating TLR9 and/or cGAS activation. Ox-mtDNA engagement of TLR9 and cGAS was confirmed in MDS specimens by IF colocalization with corresponding IFM activation, as well as in MDS somatic gene mutation murine models (Tet2, SRSF2, U2AF) vs. Wt controls. Evaluation of surface TLR9 by flow cytometry showed significantly increased membrane expression in MDS CD34+ BMMC (n=4) vs. healthy donors (n=13) (p Collectively, these data indicate that ox-mtDNA both directly engages NLRP3 and the DNA sensors TLR9/cGAS to induce IFM activation and pyroptosis, creating a feed forward inflammatory cascade that extends to neighboring cells. Ox-mtDNA may serve as a biomarker and companion diagnostic for pyroptosis execution in MDS. Disclosures List: Celgene: Research Funding.