Loss of Microtubule Protein Stathmin-1 Impairs Autophagy in Hematopoietic Stem Cells

Stathmin-1 (Stmn1) is a cytoplasmic phosphoprotein that regulates microtubule dynamics by promoting microtubule catastrophe or the sequestration of free alpha/beta-tubulin heterodimers. It is highly expressed in normal hematopoietic stem cells (HSCs), and its expression declines with cellular differentiation. Also known as oncoprotein 18 (OP18) or leukemia-associated protein 18 (LAP18), Stmn1 is overexpressed in both myeloid and lymphoid leukemias. Despite its robust expression in normal HSCs and its overexpression in leukemic cells, little is known about Stmn1's role in both normal and malignant HSCs. Herein, we have identified Stmn1 as a novel regulator of HSC function by which it regulates the autophagy/ mitophagy pathway.

To assess the role of Stmn1 in HSCs, we first determined expression levels and localization by confocal microscopy. Consistent with its role as a microtubule destabilizer, Stmn1 expression in HSCs coincides with tubulin, and Stmn1 deficient HSCs have unstable microtubule networks in preliminary data by live cell imaging. We next assessed HSC numbers and function in Stmn1^-/- mice. While Stmn1^-/- mice have similar HSC numbers to their WT counterparts, their HSCs have markedly impaired function with a significant loss of repopulating activity compared to WT HSCs in competitive transplantations (10% vs 60% chimerism at 24 weeks, p<0.0001). Similar defects in repopulating activity were seen in non-competitive transplants as well as intra-tibial transplants (to circumvent potential homing defects). Further, Stmn1^-/- mice had delayed hematopoietic recovery compared to WT controls following a single dose of 5-fluorouracil (5-FU), and Stmn1^-/- HSCs formed fewer colonies when plated in MethoCult and were not capable of serial replating. Finally, Stmn1^-/- HSCs are hyper-quiescent at baseline (but could be stimulated to cycle in response to cytokine stimulation, 3.4% vs. 7% S/G₂/M phase following stimulation), and they have elevated levels of apoptosis compared to WT (18.5% vs. 9.90%).

RNA sequencing of sorted HSCs showed alterations in genes associated with cellular metabolism, particularly OXPHOS and mTOR pathways, in the Stmn1^-/- HSCs compared to WT. Further analysis showed that Stmn1^-/- HSPCs have impaired mitochondrial maximal respiratory capacity compared to WT in the Mito stress test measured by the Seahorse analyzer (p=0.0193), and increased mtROS as assessed by a flow-based assay (p=0.009). When analyzed by TEM, Stmn1^-/- HSCs displayed mitochondria with abnormal cristae morphology (p=0.0149).

Microtubule-mitochondria associations facilitate mitophagy, a specialized form of autophagy and a major regulator of mitochondrial quality, and we next considered that this process may be impaired in the absence of Stmn1. Indeed, Stmn1^-/- HSC showed decreased autophagy with decreased expression of the autophagy-associated protein LC3 by confocal microscopy. Western blot assays similarly showed decreased levels of autophagy-associated LC3 and p62 proteins. Further, mitochondrial turnover (flux) in lysosomes was reduced in Stmn1^-/- HSCs as indicated by reduced accumulation of LC3 in the presence of the inhibitor of lysosomal proteolysis, leupeptin (p<0.05). Stmn1^-/- HSCs also showed altered localization of the mitophagy-promoting protein Parkin, and decreased Drp1 expression (indicating altered fission) by high-resolution confocal immunofluorescence. Preliminary live cell imaging data suggests that Stmn1^-/- HSCs have altered lysosome localization, further supporting that Stmn1 loss leads to impairment in the autophagy/ mitophagy pathway. Finally, we found that treatment of mice with the autophagy-inducer spermidine increased LC3 expression in the Stmn1^-/- HSCs and improved their serial replating capacity in MethoCult.

Together, these data identify Stmn1 as a critical regulator of HSC function and suggest a model whereby Stmn1 promotes normal microtubule dynamics and mitochondrial health in HSCs by regulating autophagy/ mitophagy pathways. Ongoing experiments aim to understand Stmn1's role in regulating microtubule networking and its relationship with mitochondria in both normal and malignant HSCs and elucidate the effects of Stmn1 loss on leukemic cell growth and viability, highlighting Stmn1 as a potential therapeutic target for multiple hematological malignancies

Challen:Pairidex Inc.: Current equity holder in private company, Other: Scientific Advisory Board; Incyte: Consultancy, Research Funding; Ajax Therapeutics: Consultancy, Research Funding; ReNAgade Therapeutics Management: Consultancy, Research Funding. Signer:Syndax: Current equity holder in publicly-traded company; Exact Sciences: Current equity holder in publicly-traded company, Other: Spouse employment; Vir: Current equity holder in publicly-traded company; Illumina: Current equity holder in publicly-traded company.