The Function and Mechanism Research of NLRP6 in Bone Marrow Microenvironment in Myeloproliferative Neoplasms

Since the important role of inflammation in myeloproliferative neoplasms (MPN) progression, inflammasome is an important regulator of inflammation involved in the development of tumor. Here, we aimed to investigate the function of NLRP6 in bone marrow (BM) microenvironment in MPN in the hope of getting better treatment for MPN.

We first analyzed that NLRP6 was highly expressed in BM mononuclear cells from individuals newly diagnosed with ET, PV, PMF patients (with or without JAK2 ^V617Fmutation). Similar results were obtained in BM cells and BM stromal cells of MPL ^W515L transduced C57BL/6 mice (C57BL/6 MPN). The results were validated that NLRP6 of the BM microenvironment was involved in the development of MPN.

We thus hypothesized that the absence of NLRP6 in BM microenvironment affects the development of MPN. NLRP6 ^-/- mice and C57BL/6 mice MPN models were established by the same method. The experiments demonstrated that overall survival of NLRP6 ^-/- MPN mice was significantly prolonged compared to C57BL/6 MPN mice. Loss of NLRP6 in BM microenvironment interfered with hematopoiesis of BM, but it had no effect on the extramedullary hematopoiesis (EMH) in spleen. Pathological analysis showed that the destruction of spleen structure in NLRP6 ^-/- MPN mice and the infiltration of atypical megakaryocytes were less than C57BL/6 MPN mice. The tumor load in spleen was significantly reduced and the structure of spleen was intact, the hematopoietic function of the BM returned to normal and the tumor load in the BM was significantly reduced at 5 weeks in NLRP6 ^-/- MPN mice after sequential transplantation. It suggested that knockout of NLRP6 in the BM microenvironment significantly attenuated the tumorigenicity of malignant HSCs, delayed or even inhibited the development of MPN disease.

The expression of inflammatory cytokines, abnormal megakaryocytes and the phenomenon of aggregation and reticular fibers in BM microenvironment were significantly reduced in NLRP6 ^-/- MPN group compared with the control group. After sequential transplantation, the destruction of BM structure was alleviated, megakaryocytes aggregation and heteromorphic megakaryocytes in BM were reduced, and reticular fibers were significantly diminished. Furthermore, the cytokines promoting disease progression in BM microenvironment were significantly reduced in NLRP6 ^-/- MPN group, suggesting that knockout of NLRP6 can improve the BM microenvironment and alleviate the progression of myelofibrosis.

Finally, to gain better insight into the role of NLRP6 inflammasome in MPN pathogenesis, we sought to dissect the changes with scRNA-seq and pathway identification based on available data and bioinformatic pathway analysis in NLRP6 ^-/- mice. The results of scRNA-seq analysis showed that C18 population of malignant HSCs of recipients in primary transplantation was added, C16, C13 and C9 cell populations disappeared after deletion of NLRP6 in BM microenvironment, while C5, C6, C7, C10 and C12 cell populations increased significantly. Regulon of various subpopulations of malignant HSCs changed significantly, mainly involving cell development and differentiation, immune response, inflammatory BM microenvironment, vascular endothelial homeostasis and tumor related genes.

Our results indicate that knockout of NLRP6 in the BM microenvironment may affect the progression of MPN by altering some malignant HSCs subpopulations and regulons, participating in immune, inflammatory, tumor metabolism and other signaling pathways to regulate the functional characteristics of tumor stem cells of MPN.Our data here provide new evidence for NLRP6 as potential target in the treatment of MPN.