The Role of Stathmin-1 in the Regulation of Hematopoietic Stem Cells

Stathmin-1 is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of alpha-beta tubulin heterodimers. Also known as Oncoprotein 18 (OP18), Stathmin-1 is highly expressed in a wide variety of tumors, including high-risk myelodysplastic syndromes and acute leukemias (Machado-Neto et al, 2014). Stathmin-1 is also highly expressed in normal hematopoietic stem cells (HSCs), and its loss is associated with impaired erythroid differentiation and altered megakaryopoiesis (Ramlogan-Steel et al, 2021). While these prior studies have established a role of Stathmin-1 in normal erythro-megakaryopoiesis, its role in hematopoiesis- particularly in hematopoietic stem and progenitor cells- is not clear. Given the high expression of Stathmin-1 in HSCs and the importance of microtubule dynamics to HSC differentiation, we hypothesized that Stathmin-1 loss may impact normal HSC function.

To address the role of Stathmin-1 in HSCs, we characterized HSC phenotype and function in a Stathmin-1 knockout mouse (Stmn1^-/-). Under homeostatic conditions, Stmn1^-/- HSC number and frequency are normal compared to wild-type (WT) mice. However, Stmn1^-/- HSCs have markedly impaired function, as their bone marrow (BM) failed to engraft in competitive transplants (10% vs. 60% chimerism at 24 weeks post-transplant p<0.0001). Competitive intra-tibia transplants were then done to circumvent potential homing defects, and again Stmn1^-/- BM displayed a significant engraftment defect compared to WT (10% vs. 78% chimerism at 24 weeks p<0.0001). Of note, Stmn-1^-/- HSCs performed equivalently to WT HSCs in an in vivo homing assay, further suggesting their lack of engraftment is not due to homing defects. Stmn1^-/- BM cells were capable of engraftment in non-competitive transplants, albeit less robustly than WT cells (42% vs. 90% p<0.01). Treatment of WT and Stmn1^-/- mice with one single dose of the chemotherapeutic agent 5-fluorouracil showed that Stmn1^-/-mice have slower tri-lineage recovery than their WT littermates (p<0.01 at day 15). Consistent with their reduced in vivo function, Stmn1^-/- HSCs produced fewer colonies than WT HSCs when plated in Methocult. They were also not capable of serial replating, supporting that loss of Stathmin-1 is associated with impaired HSC function.

Consistent with its known role in regulating microtubules, Stmn1^-/- HSCs displayed altered microtubule morphology by confocal microscopy with a lack of normal nuclear invaginations compared to WT HSCs. Analysis of cycling status revealed that Stmn1^-/- HSCs are more quiescent than WT HSCs at baseline (1.3% vs. 3.4% at S/G₂/M phase) and in response to 24 hours of G-CSF stimulation (3.4% vs. 7% at S/G₂/M phase). In addition, Stmn1^-/- HSCs have higher rates of apoptosis than their WT counterparts (18.5% vs. 9.90%), suggesting that Sthamin-1 loss leads to both impaired HSC cycling and increased cell death.

We next performed RNA-seq on sorted Stmn1^-/- and WT HSCs to gain further mechanistic insight into their impaired function. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function, and cell differentiation in the Stmn1^-/- HSCs compared to WT controls. Consistent with these results, measurement of mitochondrial function (oxygen consumption rate) using the Seahorse analyzer showed that Stmn1^-/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT. Together, these data suggest a model whereby Stathmin-1 regulates microtubule dynamics in HSCs to promote optimal mitochondrial function to support cellular cycling, survival and self-renewal.

Challen:Incyte: Consultancy, Other: Sponsored Research agreements.