Aging of the Vascular Niche Enhances Leukemia-Initiating Cell Metabolic Switch
Authors: Lazzari Elisa , Ramalingam Pradeep , Poulos Michael G. , Butler Jason M. 
Published: October 2019

Abstract
Bone marrow endothelial cells (BMECs) are an indispensable component for the hematopoietic stem cell (HSC) niche. In particular, Akt-activation endows ECs with the instructive capacity to support the self-renewal of HSCs to maintain homeostasis and promote the regeneration of the hematopoietic system following myeloablative stress, whereas MAPK signaling promotes HSC differentiation into lineage committed progenitors. Our group has recently demonstrated that in both in vitro and in vivo settings, aged BMECs can instruct an aged phenotype in young HSCs. Notably, we have generated preliminary data demonstrating that young BMECs maintain high levels of Akt signaling, whereas aged BMECs preferential signaling through MAPK. These data suggest that aged BMECs could be primed to negatively affect HSC function, which may lead to aged-related hematopoietic disorders, including the development of hematopoietic malignancies.
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, which incidence is predicted to rise with the expected increase in the elderly population. Disease recurrence is driven by a rare population of tumor cells, capable of self-renewal and expansion similarly to normal HSCs. These cells, referred as leukemia initiating cells (LIC), have the ability to persist during chemotherapy, and to repopulate disease. Notably, AML is known to alter the normal bone marrow vascular architecture, leading to increased vascular leakiness and to secrete a number of angiogenic factors, including VEGF-A, which has been suggested to support leukemic growth. As a consequence, the vascular niche might actively contribute to the maintenance and survival of LICs during the course of the disease and after induction of remission by chemotherapeutic agents. To this end we have generated data where we demonstrate that transplantation of AML LICs resulted in significant increases in the phosphorylation states of AKT and MAPK signaling. This data confirms that leukemic cells hijack the pro-HSC EC-specific Akt signaling pathway to enable their progression, but also over activate signaling pathways that have been demonstrated to support the differentiation of normal HSCs and support survival of AML.
To formally test if endothelial MAPK signaling impacts the ability of BMECs to support functional hematopoiesis, we generated a mouse model where mice carrying a Rosa26 Stop/Floxed MEK1DD (mutant MAPKK1 that renders MAPK constitutively activate) were crossed to a tamoxifen inducible EC-specific VEcadherin Cre transgenic mouse to generate MAPKVCC mice. Analysis of these mice revealed that MAPKVCC mice manifested a profound decline in their HSC and progenitor potential resulting from loss of vascular integrity and enhanced inflammation within their BM microenvironment. We next isolated primary BMECs from MAPKVCC mice or littermate controls, and co-cultured them in an ex vivo platform with putative MLL-AF9 AML LICs (cKit+CD34+CD11b+). We observed that MAPK pathway activation conferred BMECs the ability to significantly expand putative LICs ex vivo. Moreover, transplantation of LICs first expanded on MAPKVCC BMECs resulted in notably shorter survival of recipient mice, compared to mice receiving control BMEC-expanded LICs.
We further characterized MapkVCC BMEC metabolic profile by Seahorse XFe96 Extracellular Flux Analyzer. MapkVCC BMECs displayed a hyperglycolytic phenotype at steady state as well as increased glycolytic capacity under stress conditions, compared to control BMECs. Moreover, MapkVCC BMECs showed high respiratory capacity, thus phenocopying the metabolic profile of aged BMECs and tumor-associated ECs. Notably, MAPKVCC BMECs induced a metabolic switch in MLL-AF9 LICs ex vivo, towards increased glycolysis in both steady and stressed state and retained oxidative respiration potential under stress. These results suggest that age-associated aberrant MAPK signaling in the BM endothelial niche enhances the acquisition of the Warburg's effect in LICs, providing a selective microenvironment for the emergence of malignant clones at the expenses of normal hematopoiesis.

Disclosures
No relevant conflicts of interest to declare.