Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell malignancies that can phenotypically resemble other hematologic disorders. Thus, tools that may add to current diagnostic practices could aid in disease discrimination. Constitutive innate immune activation is a pathogenetic driver of ineffective hematopoiesis in MDS through Nod-like receptor protein 3 (NLRP3)–inflammasome-induced pyroptotic cell death. Oxidized mitochondrial DNA (ox-mtDNA) is released upon cytolysis, acts as a danger signal, and triggers inflammasome oligomerization via DNA sensors. By using immortalized bone marrow cells from murine models of common MDS somatic gene mutations and MDS primary samples, we demonstrate that ox-mtDNA is released upon pyroptosis. ox-mtDNA was significantly increased in MDS peripheral blood (PB) plasma compared with the plasma of healthy donors, and it was significantly higher in lower-risk MDS vs higher-risk MDS, consistent with the greater pyroptotic cell fraction in lower-risk patients. Furthermore, ox-mtDNA was significantly higher in MDS PB plasma compared with all other hematologic malignancies studied, with the exception of chronic lymphocytic leukemia (CLL). Receiver operating characteristic/area under the curve (ROC/AUC) analysis demonstrated that ox-mtDNA is a sensitive and specific biomarker for patients with MDS compared with healthy donors (AUC, 0.964), other hematologic malignancies excluding CLL (AUC, 0.893), and reactive conditions (AUC, 0.940). ox-mtDNA positively and significantly correlated with levels of known alarmins S100A9, S100A8, and apoptosis-associated speck-like protein containing caspase recruitment domain (CARD) specks, which provide an index of medullary pyroptosis. Collectively, these data indicate that quantifiable ox-mtDNA released into the extracellular space upon inflammasome activation serves as a biomarker for MDS and the magnitude of pyroptotic cell death.

NLRP3 inflammasome and IFN-stimulated gene (ISG) induction are key biological drivers of ineffective hematopoiesis and inflammation in myelodysplastic syndromes (MDSs). Gene mutations involving mRNA splicing and epigenetic regulatory pathways induce inflammasome activation and myeloid lineage skewing in MDSs through undefined mechanisms. Using immortalized murine hematopoietic stem and progenitor cells harboring these somatic gene mutations and primary MDS BM specimens, we showed accumulation of unresolved R-loops and micronuclei with concurrent activation of the cytosolic sensor cyclic GMP-AMP synthase. Cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling caused ISG induction, NLRP3 inflammasome activation, and maturation of the effector protease caspase-1. Deregulation of RNA polymerase III drove cytosolic R-loop generation, which upon inhibition, extinguished ISG and inflammasome response. Mechanistically, caspase-1 degraded the master erythroid transcription factor, GATA binding protein 1, provoking anemia and myeloid lineage bias that was reversed by cGAS inhibition in vitro and in Tet2 –/– hematopoietic stem and progenitor cell–transplanted mice. Together, these data identified a mechanism by which functionally distinct mutations converged upon the cGAS/STING/NLRP3 axis in MDS, directing ISG induction, pyroptosis, and myeloid lineage skewing.

To better understand the signaling complexity of AXL, a member of the tumor-associated macrophage (TAM) receptor tyrosine kinase family, we created a physical and functional map of AXL signaling interactions, phosphorylation events, and target-engagement of three AXL tyrosine kinase inhibitors (TKI). We assessed AXL protein complexes using proximity-dependent biotinylation (BioID), effects of AXL TKI on global phosphoproteins using mass spectrometry, and target engagement of AXL TKI using activity-based protein profiling. BioID identifies AXL-interacting proteins that are mostly involved in cell adhesion/migration. Global phosphoproteomics show that AXL inhibition decreases phosphorylation of peptides involved in phosphatidylinositol-mediated signaling and cell adhesion/migration. Comparison of three AXL inhibitors reveals that TKI RXDX-106 inhibits pAXL, pAKT, and migration/invasion of these cells without reducing their viability, while bemcentinib exerts AXL-independent phenotypic effects on viability. Proteomic characterization of these TKIs demonstrates that they inhibit diverse targets in addition to AXL, with bemcentinib having the most off-targets. AXL and EGFR TKI cotreatment did not reverse resistance in cell line models of erlotinib resistance. However, a unique vulnerability was identified in one resistant clone, wherein combination of bemcentinib and erlotinib inhibited cell viability and signaling. We also show that AXL is overexpressed in approximately 30% to 40% of nonsmall but rarely in small cell lung cancer. Cell lines have a wide range of AXL expression, with basal activation detected rarely. Implications: Our study defines mechanisms of action of AXL in lung cancers which can be used to establish assays to measure drug targetable active AXL complexes in patient tissues and inform the strategy for targeting it's signaling as an anticancer therapy.

Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS.

Myelodysplastic Syndromes (MDS) are 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). Toll-like receptor 9 (TLR9) is an endosomal, DNA sensing pattern recognition receptor that primes and activates the IFM and ISG response through myddosome signaling upon engagement by hypomethylated, CpG-rich DNA. Oxidized newly synthesized mitochondrial DNA (ox-mtDNA) is released into the cytosol upon TLR/IL-1R activation to trigger Nlrp3 IFM activation. Upon lytic pyroptotic cell death, however, ox-mtDNA is released into the extracellular space. We previously reported that concentrations of ox-mtDNA, a native TLR9 ligand, are profoundly increased in MDS patient plasma compared to age-matched controls and other hematologic malignancies (Ward G, et. al. ASH 2018). The aim of this investigation was to determine if ox-mtDNA acts as a danger associated molecular pattern (DAMP) to propagate the inflammatory response and IFM activation in neighboring cells through TLR9. We have shown that MDS hematopoietic stem and progenitor cells (HSPC) redistribute TLR9 to display cell surface TLR9 expression. We hypothesized that this increased surface expression is induced in response to ox-mtDNA in the BM plasma. To test this, SKM1 and U937 cells were incubated for 2 hours with 50ng/mL ox-mtDNA (ND1 gene, unmethylated, amplified with oxidized guanosine) and TLR9 expression was assessed by flow cytometry (FC). Following incubation, both cell lines significantly increased TLR9 surface expression (n=3, p<0.03).We next confirmed that TLR9 interacts with ox-mtDNA by immunofluorescence (IF) microscopy in MDS samples and murine somatic gene mutation (SGM) models (Tet2-/- and Srsf2P95H). We further investigated the relationship between TLR9 and ox-mtDNA during IFM activation. Treatment of SKM1 and U937 cells with the TLR4 ligand LPS and priming agents ATP/nigericin initiates a robust increase in ox-mtDNA that co-localized intracellularly with TLR9 by IF microscopy. Furthermore, upon incubation with ox-mtDNA, ox-mtDNA bound TLR9 is internalized. Additionally, interferon regulatory factor 7 (IRF7), an ISG transcription factor induced by TLR9, translocates to the nucleus following treatment, confirming TLR9 activation in response to ox-mtDNA. We next assessed whether IFM activation by ox-mtDNA is TLR9-dependent. 50ng/mL ox-mtDNA was incubated with 3 leukemic cell lines: SKM1, U937, and THP1 that display varying levels of endogenous TLR9 expression, as well as corresponding TLR9-KO cells generated by CRISPR-Cas9 editing. We found that TLR9 was necessary for IFM initiated caspase-1 cleavage in response to ox-mtDNA, thereby demonstrating the specificity of ox-mtDNA for the TLR9 sensor. Notably, receptor density determined response tempo. SKM1 cells, which have the highest TLR9 receptor density, responded within 1 hour, U937 cells which have an intermediate receptor density respond in 2 hours, while THP1 cells, which do not express endogenous TLR9 never responded to ox-mtDNA treatment. TLR9 knockout abrogated IFM activation in response to ox-mtDNA as demonstrated by caspase-1 glo ® assay (n=5, p<0.03). By western blot, TLR9 knockout cells no longer demonstrate phosphorylated NFk-B, cleaved caspase-1, and cleaved IL-1β in response to ox-mtDNA treatment. We conclude that MDS HSPC display functionally competent TLR9 on the plasma membrane which primes them for response to ox-mtDNA released by neighboring pyroptotic cells. Blocking TLR9 activation may prove to be a novel therapeutic strategy for MDS and suppression of inflammatory BM failure. Disclosures Hou: Celgene: Research Funding; Abbvie, Astellas, BMS, Celgene, Chugai, Daiichi Sankyo, IQVIA, Johnson & Johnson, Kirin, Merck Sharp & Dohme, Novartis, Pfizer, PharmaEssential, Roche, Takeda: Honoraria. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.