Hematopoiesis Inhibition In Juvenile Myelomonocytic Leukemia And The Underlying Mechanism

Background:Juvenile myelomonocytic leukemia（JMML）is an aggressive childhood myeloproliferative disorder or neoplasms（MPD/MPN）.Hematopoiesis inhibition is always observed during JMML progression,which disrupts the integrity of immune system.The high risk of severe infection or other related complications,which result from hematopoiesis inhibition,is related to poor prognosis.However,the underlying mechanism of hematopoiesis inhibition in JMML remains largely unknown.Gain-of-function mutations in Ptpn11 are found in 35%of JMML patients.The most common and active Ptpn11^E76K mutations would aberrantly stimulate RAS signaling,leading to JMML.In this study,Ptpn11^E76K mutant mice were used as JMML model to describe the characteristics of hematopoiesis alterations in JMML,and to figure out the underlying mechanism.Objects:To investigate the underlying mechanism of hematopoiesis inhibition in JMML.Methods:Generated myeloid specific Ptpn11^E76K mutant mice as the animal model of JMML disease.The characteristics of JMML in model mice were verified by flow cytometry combined with histopathology.Phenotypic verification:The percentages,spatial distribution and functions of hematopoietic stem cells（HSCs）in the bone marrow（BM）of mice with different genotypes were determined and compared by flow cytometry and tissue immunofluorescence staining.Mixed transplantation assay was processed,and hematopoietic cell reconstitution,HSC pool size,and HSC cycling status in the recipients were determined by flow cytometry.Mechanism verification:Chemokine-cytokine array was used to identify the production of inflammatory cytokines in the BM plasma from mice with different genotypes.Gene knockout model of related cytokine receptor was constructed,and myeloid-biased hematopoiesis of HSCs from mice with different genotypes was compared in mouse model or in in-vitro culture assay.Clinical samples:The characteristics of cytokine expression in JMML patients’samples was determined by Cytometric Bead Array（CBA）assay,and the capability of inducing myelo-hematopoiesis was evaluated by co-culture assay.Results:1.Hematopoiesis inhibition in JMML mouse model:We induced the Ptpn11^E76K mutation,the most common mutation identified in JMML,specifically in the myeloid lineage with hematopoietic stem cells（HSCs）spared.These mice uniformly developed profound JMML-like MPN.Importantly,HSCs in the same BM microenvironment were aberrantly activated and differentiated at the expense of self-renewal.2.JMML tumor cells play a key role in inducing hematopoiesis inhibition:A similar phenomenon,that HSCs were aberrantly hyperactivated,was observed in wild-type（WT）donor HSCs when co-transplanted with Ptpn11^E76K/+BM cells into WT mice.3.The underlying mechanism of hematopoiesis inhibition induced by JMML tumor cells:（1）Cytokine profiling revealed that Ptpn11^E76K/+MPN cells highly produced IL-1β,but not IL-6,TNF-α,IFN-γ,IL-1α,or other inflammatory cytokines.（2）Depletion of the IL-1R effectively restored stem cell quiescence and normalized their pool size,rescuing HSCs from exhaustion in Ptpn11^E76K/+/IL-1R^-/-double mutant mice.（3）Co-culture testing showed that JMML/MPN cells robustly accelerated differentiation in hematopoietic stem/progenitor cells,which can be rescued by IL-1βreceptor（IL-1R）deficiency.4.BM sample from JMML patient produced higher level of IL-1β,and accelerated the myeloid differentiation of Cord-blood stem cells.Conclusion:JMML tumor cells disrupt normal hematopoiesis by imposing inflammatory stress through overproduction of IL-1β.