Leukemic Stem Cells Co-Opt Normal Bone Marrow Niches As a Source of Energy and Antioxidant Defence
Authors: Forte Dorian , García-Fernández María , Sánchez-Aguilera Abel , Stavropoulou Vaya , Fielding Claire , Martín-Pérez Daniel , Tzankov Alexandar , Schwaller Juerg , Méndez-Ferrer Simón 
Published: April 2021

Abstract
Modulation of oxidative stress as well as metabolic adjustments are key aspects in cancer stem cell-derived diseases, such as acute myelogenous leukemia (AML). Metabolic reprogramming in leukemia seems to be influenced by the crosstalk of malignant cells with their surrounding microenvironment. Reactive oxygen species (ROS), mainly generated by the mitochondria, are relatively high in malignant cells, without reaching a cytotoxic level that would compromise their survival. In addition, chemotherapy efficacy lies on increased ROS-mediated cellular damage. Cumulative evidence shows that AML often exhibits poor response to chemotherapy partly due to the support of leukemic stem cells (LSCs) by the bone marrow (BM) microenvironment, which includes BM mesenchymal stem cells (BMSCs). However, how exactly this happens, i.e. the specific mechanism(s) and cell populations implicated, remains incompletely understood.
Hematopoietic stem cell (HSC)-niche forming BMSCs can be identified by the expression of the intermediate filament protein nestin and can be isolated and propagated as non-adherent 'mesenspheres', which can self-renew and support HSCs ex vivo.
Here we have studied the contribution of nestin+ BMSCs to MLL-AF9-driven AML development and resistance.
Immunohistochemistry for nestin on BM samples from AML patients (N=35) showed that the number of nestin+ niches per square millimeter was 2.4-fold higher as compared to control samples (N=12). To study the role of BMSCs in AML, we used a doxycycline-inducible iMLL-AF9 mouse strain that closely mimics the human disease (Stavropoulou V et al., Cancer Cell 2016;30:43-58). Development of AML in iMLL-AF9 mice caused a significant reduction of stromal cells in the BM but it did not reduce the number of BM nestin+ cells. To study the possible contribution of nestin+ cells to leukemia development, we partially depleted nestin+ cells by inducing diphtheria toxin expression in these cells using Nestin-creERT2 ; iDTA mice previously transplanted in a competitive fashion with pre-leukemic and normal BM cells. Nestin+ cell depletion selectively diminished leukemia burden in BM, spleen and blood, and reduced the number of primitive leukemic cells, without affecting normal residual hematopoiesis. Combined nestin+ cell depletion and standard chemotherapy further exaggerated the elimination of BM leukemic cells, suggesting that nestin+ cells contribute to chemotherapy resistance.
To investigate the crosstalk of BMSCs and leukemic blasts, we developed a novel in vitro co-culture system using BM mesenspheres and iMLL-AF9+ leukemic blasts. Co-cultures with nestin+ mesenspheres promoted leukemic blast survival under metabolic stress (FBS deprivation) and chemotherapy (cytarabine, Ara-C). Increased survival correlated with a strong protection against excessive ROS levels and lipid peroxidation. Particularly, reduced glutathione (GSH), a key antioxidant element, increased in leukemic blasts co-cultured with nestin+ mesenspheres.
Mitochondrial transfer from BMSCs has also recently been proposed as a metabolic-related mechanism contributing to chemoresistance in AML (Moschoi R et al. Blood 2016;128:253-64). Using the Seahorse Extracellular Flux Analyzer, we actually revealed that leukemic blast bioenergetics increased in co-culture with mesenspheres. Surprisingly, we found that chemotherapy-induced transfer of mitochondria from BMSCs to AML cells correlated with decreased ROS levels in the leukemic blasts, pointing out mitochondria transfer as an important ROS detoxifying mechanism to allow chemoresistance. We demonstrated that cell-to-cell contact could be required for Ara-C-mediated protective effects of mesenspheres on leukemic blasts and their mitochondria transfer. Accordingly, either the inhibition of GSH generation or mitochondrial transfer (by avoiding cell contact) may reduce BMSCs survival support after chemotherapy.
Altogether, these results suggest that simultaneous co-opting of energy sources and anti-oxidant mechanisms of BMSCs contribute to chemoresistance in AML.

Disclosures
No relevant conflicts of interest to declare.