Concerted roles of PTEN and ATM in controlling hematopoietic stem cell fitness and dormancy
Jerome Fortin, Christian Bassi, Parameswaran Ramachandran, Wanda Y. Li, Ruxiao Tian, Ida Zarrabi, Graham Hill, Bryan E. Snow, Jillian Haight, Chantal Tobin, Kelsey Hodgson, Andrew Wakeham, Vuk Stambolic, Tak W. Mak
Jerome Fortin, Christian Bassi, Parameswaran Ramachandran, Wanda Y. Li, Ruxiao Tian, Ida Zarrabi, Graham Hill, Bryan E. Snow, Jillian Haight, Chantal Tobin, Kelsey Hodgson, Andrew Wakeham, Vuk Stambolic, Tak W. Mak
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Research Article Hematology

Concerted roles of PTEN and ATM in controlling hematopoietic stem cell fitness and dormancy

Abstract

In order to sustain proficient life-long hematopoiesis, hematopoietic stem cells (HSCs) must possess robust mechanisms to preserve their quiescence and genome integrity. DNA-damaging stress can perturb HSC homeostasis by affecting their survival, self-renewal, and differentiation. Ablation of the kinase ataxia telangiectasia mutated (ATM), a master regulator of the DNA damage response, impairs HSC fitness. Paradoxically, we show here that loss of a single allele of Atm enhances HSC functionality in mice. To explain this observation, we explored a possible link between ATM and the tumor suppressor phosphatase and tensin homolog (PTEN), which also regulates HSC function. We generated and analyzed a knockin mouse line (PtenS398A/S398A), in which PTEN cannot be phosphorylated by ATM. Similar to Atm+/–, PtenS398A/S398A HSCs have enhanced hematopoietic reconstitution ability, accompanied by resistance to apoptosis induced by genotoxic stress. Single-cell transcriptomic analyses and functional assays revealed that dormant PtenS398A/S398A HSCs aberrantly tolerate elevated mitochondrial activity and the accumulation of reactive oxygen species, which are normally associated with HSC priming for self-renewal or differentiation. Our results unveil a molecular connection between ATM and PTEN, which couples the response to genotoxic stress and dormancy in HSCs.

Authors

Jerome Fortin, Christian Bassi, Parameswaran Ramachandran, Wanda Y. Li, Ruxiao Tian, Ida Zarrabi, Graham Hill, Bryan E. Snow, Jillian Haight, Chantal Tobin, Kelsey Hodgson, Andrew Wakeham, Vuk Stambolic, Tak W. Mak

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Figure 6

PtenS398A/S398A hematopoietic stem cells tolerate elevated levels of oxidative stress.

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PtenS398A/S398A hematopoietic stem cells tolerate elevated levels of ox...
(A) Flow cytometry plots depicting the gating strategy to identify Lin–cKit+Sca1– cells (LK), Lin–cKit+Sca1+ cells (LSK), long-term hematopoietic stem cells (LT-HSCs), multipotent progenitors (MPPs), 2 subsets of hematopoietic progenitors (HPC-1 and HPC-2), and PROCR+ LT-HSCs in Pten+/+ and PtenS398A/S398A mice. The parent populations are indicated on top of the plots, and the antibodies used are indicated on the axes. In the PROCR staining histogram, blue lines indicate cells from Pten+/+ mice, and orange lines indicate cells from PtenS398A/S398A littermates. Quantification of LT-HSCs expressing PROCR is shown at the right. Each symbol represents an individual animal. (B) Quantification of LT-HSCs expressing PROCR in Atm+/+, Atm+/–, and Atm–/– littermates. Each symbol represents an individual mouse. (C) Flow cytometry plots (left) and quantification (right) of fluorescence intensity for dihydrorhodamine (DHR), an indicator of reactive oxygen species (ROS) (left) and for MitoTracker Green, an indicator of mitochondrial content (right) in PROCR+ LT-HSCs in Pten+/+ and PtenS398A/S398A mice. Each symbol represents an individual animal. Gray traces show the background fluorescence intensity in the absence of the DHR and MitoTracker dyes. (D) Relative levels of mitochondrial over nuclear DNA, assessed by qPCR measurements of Cytb and Actb, respectively, in sorted PROCR+ LT-HSCs from Pten+/+ and PtenS398A/S398A mice. Each symbol represents data from cells isolated from an individual mouse. In all panels, mean and SEM are shown. *P < 0.05, **P < 0.01, ***P < 0.001, assessed by t test (A, C, and D) or 1-way ANOVA with Tukey’s multiple-comparison test (B).