NOX2 Derived Superoxide Induces Pro-Tumoral p16INK4a Driven Senescence in the AML Bone Marrow Microenvironment
Authors: Hellmich Charlotte , Abdul-Aziz Amina , Sun Yu , Marlein Christopher Richard , Forde Eoghan , Piddock Rachel E , Shafat Manar , Morfakis Adam , Tarang Mehta , Di Palma Federica , Ingham Christopher , Collins Angela , Haestier Anna , Campisi Judith , Bowles Kristian , Rushworth Stuart 
Published: October 2019

Abstract
Background
Acute myeloid leukaemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. Senescence is the irreversible arrest of cell proliferation and is associated with the senescence associated secretary phenotype (SASP). The senescent phenotype has been associated with pro-tumoral changes that support proliferation and survival of malignant cells. Because AML is an age-related disease and highly dependent on the BM microenvironment, which naturally becomes senescent with age, we hypothesized, and subsequently tested the idea, that AML not only favors a senescent microenvironment but might actively shape a senescent microenvironment to promote tumor proliferation and survival.
Methods
Primary AML and bone marrow stroma was isolated from bone marrow samples obtained from patients following informed consent and non-malignant CD34+ cells were isolated from cord blood following informed consent. All primary tissue was used under approval from the UK Health Research Authority. NSG, p16-3MR(Demaria et al. 2014 Cell Development) and wildtype C57Bl/6 animals were used and experiments were conducted with approval from the UK Home Office and University of East Anglia Animal Welfare and Ethical Review Board. Primary AML and non-malignant human CD34+ cells were engrafted into NSG mice (AML-NSG and hu-NSG). Bone marrow stromal cells (BMSCs) were isolated by cell sorting for mouse CD45-, Ter119-, CD31-, CD105+, CD140a+. Syngeneic AML cells were generated from lineage negative cells transduced with the oncogene MN1. Senescence was determined by p16, p21 mRNA expression, SASP expression (IL-6 and MIP3a), senescence associated β-Galactosidase activity and using themonomeric red fluorescent protein (mRFP) component of the p16-3MR mouse model. Oxidative stress and superoxide levels were measured using 2',7'-dichlorofluorescein (DCF) fluorescence and the Amplex Red assay. NOX-2 in AML was inhibited by RNAi.
Results
First, we isolated and analysed BMSC from AML-NSG and hu-NSG. Using real-time PCR we detected high expression of p16 and p21 in the BMSC isolated from AML-NSG but not hu-NSG. Moreover, we detected high mouse IL-6 and MIP3a cytokine levels from serum isolated from AML-NSG but not hu-NSG. MN1 engrafted into the p16-3MR model induced mRFP expression in the BMSC population. This shows that in both a xenograft and syngeneic model of AML the BMSC population becomes senescent. Using the p16-3MR model we selectively depleted senescent BMSC via the viral thymidine kinase death switch. Treatment of p16-3MR mice engrafted with MN1 with ganciclovir reduced tumour volume and prolonged animal survival. Since hydrogen peroxide has been shown to induce senescence we hypothesised that NOX2 derived superoxide from AML drives the BMSC senescence. BMSCs isolated from the MN1 engrafted p16-3MR model isolated BMSC show increased DCF fluorescence and isolated BM shows elevated superoxide compared to BMSC and BM isolated from control mice. NOX2 knock down resulted in significant reduction in BM derived superoxide and BMSC DCF fluorescence and this resulted in a reduced BMSC senescent phenotype.
Conclusion
Here we show that in AML, NOX2 derived superoxide induces a p16INK4a drivensenescent phenotype in the BMSC. Moreover, we find that this process forms a fundamental part of the pro-tumoral pathophysiology of the disease. The senescent bone marrow microenvironment may therefore present new treatment targets in AML.

Disclosures
No relevant conflicts of interest to declare.