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<0.05), suggesting priming for enhanced sensitivity to ox-mtDNA. Concentrations of ox-mtDNA in supernatants from THP1 (N=5, p=0.0547), U937 (N=3, p=0.0637), and SKM1 (N=3, p=0.028) cells confirmed extracellular release after LPS/ATP/Nigericin induced pyroptosis. We then investigated ox-mtDNA concentrations in peripheral blood plasma from MDS patients by ELISA. Glucose adjusted, ox-mtDNA levels were significantly increased in 177 MDS cases compared to 29 healthy donors (p<0.0001), which was validated in an independent cohort of 154 MDS cases and 34 healthy donors (p<0.0001). Ox-mtDNA was higher in lower risk (LR) compared to higher risk MDS (p<0.0001) consistent with greater pyroptotic cell fraction in LR-MDS. Further, we found a direct correlation between ox-mtDNA (n=134) and known MDS IFM-activating alarmins S100A9 (r=0.395), S100A8 (r=0.618), and percentage of ASC specks (r=0.589) (p<0.0001 for all), demonstrating specificity of ox-mtDNA as a biomarker for the magnitude of medullary pyroptosis in MDS. Importantly, compared to other hematologic malignancies, we found that plasma ox-mtDNA was significantly increased in LR-MDS (n=162) compared to ALL (n=7), de novo (n=20) and secondary AML (n=18), CML (n=8), CMML (n=18), LGL (n=19), MF (n=12), ET (n=20), PV (n=20), MM (n=18), and type 2 diabetes (n=25) where IFM activation promotes insulin resistance (p ≤0.036). There was no significant difference in oxDNA concentrations between MDS and CLL (n=50), which is reported to have high ox-mtDNA levels associated with genomic abnormalities (Collado, 2014, Biomed Res Int.). ROC/AUC analysis demonstrated that ox-mtDNA is an MDS pyroptosis-specific biomarker when compared to healthy donors (AUC=0.929). Similarly, a 5-fold cross-validation (k=5) repeated 30 times to compare MDS cases to non-MDS hematological malignancies (excluding CLL), similarly confirmed biomarker specificity (AUC=0.877).

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.