Mlkl Mediates Age-Related Attrition of Hematopoietic Stem Cells and Ineffective Hematopoiesis in Myelodysplastic Syndrome

Aging causes anemia, perturbed immunity, and increased propensity to age-related hematological diseases such as myelodysplastic syndrome (MDS). Age-dependent functional decline in hematopoietic stem cells (HSCs), which is accompanied by morphological and functional changes in their organelles as well as sterile inflammation in the bone marrow (BM), is one of the main reasons why the hematopoietic system ages. However, molecular mechanisms by which HSCs alter their function during aging remain unclear. Necroptosis is a form of programmed necrosis that underlies sterile inflammation and can take place upon exposure to inflammatory stimuli in apoptosis-resistant cells including HSCs. MLKL, the effector molecule of necroptosis that can form channel-like structure on lipid bilayers upon activation, not only induces cell death but also injures membrane-bound organelles such as mitochondria, though its significance in HSC aging remains elusive.

Our quantitative RT-PCR revealed abundant expression of MLKL in murine BM immature hematopoietic cells including HSCs (Lin^-/cKit⁺/Sca1⁺/Flk2^-/CD48^-/CD150⁺), which was validated by reanalysis of the published proteome datasets (Zaro et al., eLife 2020). Using FRET-based necroptosis reporter mice and Western blotting analysis, we found that while MLKL was inactivated at the steady state, it was readily activated in HSCs upon poly I:C injection. To study the effect of MLKL activation on HSCs, we assessed cell death of HSCs isolated from poly I:C-treated MLKL-deficient (Mlkl^-/-) mice. Unexpectedly, Annexin V/DAPI staining revealed no changes in the frequencies of dying and dead cells between wild-type (WT) and Mlkl^-/- HSCs. Nonetheless, PVA-based HSC culture revealed that the growth suppression observed in poly I:C-treated WT HSCs largely depended on MLKL (p=0.0003). Furthermore, HSC transplantation assay revealed that reduced engraftment and skewed myeloid differentiation were observed in poly I:C-treated WT HSCs but largely alleviated by MLKL deficiency (p=0.011). These results indicate that inflammation-induced MLKL activation does not cause HSC death but impairs their growth and lymphopoietic potential.

To assess the relevance of MLKL activation in HSC aging, we treated WT and Mlkl^-/- mice with serial rounds of 5-FU treatment, which were shown to induce age-related alterations in HSCs (Beerman et al., Cell Stem Cell 2013). Of note, we observed marked attenuation of age-related features such as myeloid-skewed hematopoiesis and expansion of phenotypic HSCs in 5-FU-treated Mlkl^-/- mice (p=0.0051). HSC transplantation experiments further revealed that MLKL deficiency alleviated 5-FU-induced decrease in engraftment and lymphopoietic potential of HSCs (p=0.013). Indeed, analysis of 18-month-old WT and Mlkl^-/- mice demonstrated that MLKL deficiency largely rescued myeloid-skewed hematopoiesis and loss of BM CLPs (Lin^-/cKit^lo/Sca1^lo/Flk2⁺/IL-7Rα⁺) (p=0.0047), and serial HSC transplantation revealed better self-renewal and lymphopoietic potential of aged Mlkl^-/- HSCs (p=0.0055). Importantly, multiplex ELISA assay using BM supernatants revealed no changes in concentrations of inflammatory cytokines, indicating that the observed effects in Mlkl^-/- HSCs were likely caused by cell intrinsic changes. In line with this, electron microscopy analysis revealed that age-related changes in mitochondria, such as elongated and swollen structure with disorganized cristae, were significantly attenuated in aged Mlkl^-/- HSCs (p<0.0001). JC-1 dye staining further revealed that age-related accumulation of damaged mitochondria was significantly attenuated in Mlkl^-/- HSCs (p=0.049). Finally, to evaluate the effect of MLKL on development of age-related hematological disease, we established MDS mouse models by transplantation of RUNX1^S291fs-transduced WT and Mlkl^-/- HSCs. We found that WT MDS mice mainly died of severe anemia due to ineffective hematopoiesis, whereas Mlkl^-/- MDS mice showed milder anemia and better overall survival (p=0.044), indicating that MLKL promotes ineffective hematopoiesis in MDS.

Taken together, our results identify MLKL as a key mediator of age-related functional decline in HSCs and ineffective hematopoiesis in MDS. They also suggest that pharmacological inhibition of MLKL activity is a promising strategy to better control aging of the hematopoietic system and age-related hematological disease.

Iwama:Nissan Chemical Indistries: Research Funding; Daiichi-Sankyo Pharmatheutical: Research Funding.