The NLRC4 Inflammasome Drives Myelodysplastic Syndrome By Linking Epigenetic Reprogramming and Innate Immune Signaling

Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell (HSC) malignancies characterized by dysplastic and ineffective hematopoiesis. Despite emerging evidence that chronic inflammation contributes to the development of MDS, the mechanisms underlying the dysregulation of innate immune and inflammatory signaling pathways in hematopoietic stem and progenitor cells (HSPCs) remain poorly characterized. We have used multiple MDS mouse models to identify a tumor suppressive role for the EP300 epigenetic histone acetyltransferase enzyme in the progression of MDS to AML. Based on the gene expression changes we observed, we have used one specific mouse model, that lacks Tet2 expression, to define the role that chronic inflammation plays in promoting MDS development and the mechanisms involved.

Methods We have conducted a series of in vitro, in vivo, and in silico experiments using Tet2-deficient mice, and chronic, low-dose LPS treatment to model chronic inflammation. Cytokine array immunoassays were performed to evaluate the inflammatory response of WT or Tet2-deficient mice to LPS treatment. Chimeric mice transplanted with bone marrow (BM) cells isolated from LPS or vehicle (PBS) treated WT or Tet2-deficient mice were monitored for the development of MDS and survival. Serial competitive bone marrow transplantation assays and flow cytometry profiling were used to determine the competitive advantage of LPS or PBS-treated BM cells, and their differentiation. Bioinformatic analyses of RNA sequencing (RNA-seq) results from our mouse model were performed to characterize the transcriptional profiling and identify novel innate immune pathways. Colony-forming unit (CFU) and qPCR assays were used to evaluate HSPC proliferative capacity, self-renewal, and gene expression changes.

Results We find that mice lacking Tet2 develop a hyper-inflammatory status, marked by the expression of a number of pro-inflammatory cytokines, such as Il-1b and Il-6, which are further fueled by LPS-induced chronic inflammation. Chimeric mice transplanted with BM cells isolated from LPS-treated Tet2-deficient mice exhibit a significantly shorter life span and early onset of MDS phenotypes, including dysplastic hematopoiesis and thrombocytopenia, compared to age-matched chimeric mice transplanted with BM cells isolated from PBS-treated Tet2-deficient mice or LPS-treated WT mice. Chronic inflammation promotes the self-renewal and myeloid differentiation of Tet2-deficient HSPCs in the short term; however, it accelerates HSC exhaustion in the long run. Transcription profiling and subsequent bioinformatic analyses identify functional clusters in LPS-treated Tet2-deficient HSPCs, including genes involved in the regulation of epigenetics, immunity, metabolism, and cell cycle. Detailed inspection of the most highly differentially expressed genes reveals that many genes in the Nlrc4 inflammasome pathway are upregulated in LPS-treated Tet2-deficient HSPCs. We validate changes in the expression of these genes by qPCR, and then show that deleting Nlrc4 suppresses the colony formation of Tet2-deficient HSPCs in vitro. Ongoing work focuses on evaluating the effects of deleting Nlrc4 or blocking Nlrc4 function using a small molecule inhibitor on MDS development in vivo.

Conclusions The combinatory role of intrinsic and extrinsic factors drives the development of MDS in this Tet2-deficient MDS mouse model. It is likely that different genetic mutations generate unique sensitivity to certain signaling pathways, and we demonstrate the epigenetic rewiring and activation of the Nlrc4 inflammasome pathway as a mechanistic basis for the inflammatory component of MDS [or clonal hematopoiesis of indeterminate potential (CHIP)] pathogenesis in Tet2-deficient cells. Our work has the potential to provide novel targeting opportunities in MDS management and confirm the importance of different components of inflammasome signaling in MDS.

No relevant conflicts of interest to declare.