Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Kenneth Wright, Ph.D.

Committee Member

John Cleveland, Ph.D.

Committee Member

Eric Padron, M.D.

Committee Member

Eric Lau, Ph.D.

Committee Member

Guillermo Garcia-Manero, M.D.

Keywords

DAMP, Inflammasome, Pyroptosis, Toll-like Receptor 9, Bone Marrow Failure, Caspase-1

Abstract

Myelodysplastic Syndromes (MDS) are heterogeneous bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, NLRP3 inflammasome (IFM) driven pyroptotic cell death, and the induction of interferon-stimulated genes (ISG). MDS often diagnostically resembles other hematologic disorders, as such, additional tools are needed for the discrimination from overlapping disorders. We observed that oxidized mitochondrial DNA (ox-mtDNA) is released upon cytolysis. We hypothesize that levels of ox-mtDNA are high in MDS, allowing us to use it as a diagnostic biomarker; and that cell free ox-mtDNA is contributing to feedforward BM failure.

ROC/AUC analysis demonstrated that ox-mtDNA is a sensitive and specific biomarker for MDS compared to healthy donors (AUC=0.964), and other hematological malignancies excluding CLL (AUC=0.893). Ox-mtDNA positively and significantly correlated with levels of known alarmins S100A9, S100A8, and ASC specks demonstrating utility as a biomarker for the magnitude of medullary pyroptosis in MDS.

Toll-like receptor 9 (TLR9) is an endosomal, DNA sensing pattern recognition receptor that activates the IFM and ISG response through MyDDosome signaling. We found that MDS hematopoietic stem cells have increased TLR9 cell surface expression, which is induced by exposure to ox-mtDNA. We observed ox-mtDNA treatment phenocopies MDS via caspase-1 and IL-1β cleavage, ASC specks, LDH release, and activation of ISGs. We next assessed whether IFM activation by ox-mtDNA is TLR9-dependent and found that TLR9-KO cells were no longer responsive to ox-mtDNA while TLR9 overexpression induced sensitization to ox-mtDNA. We evaluated the impact of ox-mtDNA on hematopoietic colony forming capacity (CFC) and found that normal BM CFC was diminished by ox-mtDNA and rescued by TLR9 lentiviral knockdown. Upon chemical inhibition of the TLR9 pathway, including a competitive TLR9-IgG sponge, we observed significantly decreased inflammasome and restored CFC of MDS primary samples.

We conclude that MDS BM cells have TLR9 on the plasma membrane which primes them for response to ox-mtDNA released by neighboring pyroptotic cells. Our research shows that ox-mtDNA is a novel alarmin that activated the IFM via TLR9 and provides an index of pyroptosis in MDS. Blocking TLR9 activation may provide a novel therapeutic strategy for MDS and suppression of inflammatory BM failure.

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