Relationship between m-TOR-Mediated Upregulation of Glycolysis, Chemoresistance, and Prognosis in Patients with ALL.
Authors: Buglio Daniela , Konopleva Marina , McQueen Teresa , Samudio Ismael , Broglio Kristine R. , Ruvolo Vivian , Contractor Rooha , Chen Wenjing , Kantarjian Hagop , Andreeff Michael , Weiser Mary A. 
Published: April 2020

Abstract
Background: The serine/threonine kinase AKT, a major downstream PI3K target, promotes continued cell growth and metabolism by increasing glucose uptake, stimulating glycolysis and ATP production. It has been proposed that cancer cells with a high level of constitutively active AKT depend on glucose for survival. Recent data in transgenic models demonstrated that AKT activation results in mTOR dependent transcriptional upregulation of the glycolytic enzyne HK2 and glucose transporter Glut1 via induction of HIF1-α (Nature Med2004;10:594). The purpose of this study was to determine the frequency and prognostic significance of increased expression of HK2 in patients with ALL, to determine if proglycolytic conditions, high glucose (HG) or hypoxia, promote chemoresistance in ALL blast cells, and if chemoresistance is affected by mTOR inhibition.
Methods: HK2 mRNA expression was measured by quantitative Taqman PCR in 28 primary ALL samples (14 newly diagnosed and 14 relapsed). Primary bone marrow samples (N=5) were co-cultured on either MS-5 or HS-5 stroma supplemented with 4 or 14 mM glucose for 24–48 hours. Jurkat cells were cultured in serum-free medium supplemented with 4 or 14 mM glucose under hypoxic (3% O2) or normoxic (21% O2) conditions. All samples were treated with doxorubicin (DOX - 25–50 ng/mL) +/− RAD001 (10–20 nM) or CCI779 (2.5–5 mg/mL).
Results: Increased expression of HK2 (≥ 2-fold relative to normal samples) occurred in 5/28 samples (18%). Patients with HK2 levels above the median (0.43) had a worse failure free survival (FFS) (HR 2.95; CI 0.89–9.81; P=0.08) and a significantly worse overall survival (OS) (HR 5.01; CI 1.21–20.7; P=0.03). In 4/5 (80%) of primary ALL samples, exposure to HG (14 mM) resulted in decreased apoptosis with DOX compared to normal glucose (4 mM) (mean % Ann+ 32.0 vs 42.0; P=0.047). This effect was reversed with addition of RAD001 or CCI779 (mean % Ann+ 49.36 vs 54.02; P=0.11). Jurkat cells cultured under hypoxic conditions and treated with DOX demonstrated decreased apoptosis and increased cell viability compared to normoxic conditions [mean % Ann (+) cells 39.85 (Hypoxic) vs 55.00 (Normoxic); P=0.016). This effect was reversed with addition of RAD001 under hypoxic conditions [mean % Ann (+) cells 39.85 (DOX) vs 51.92 (DOX+RAD); P=0.009] in association with decreased expression of HK2 mRNA (RE to DMSO mean 62 vs 47; P=0.26).
Conclusions: Overexpression of HK2 is associated with worse OS in pts with newly diagnosed or relapsed ALL. Exposure of ALL blast cells to high glucose or hypoxic environment results in chemoresistance to DOX. This effect is abrogated by mTOR inhibition in association with decreased expression of HK2 mRNA, suggesting that upregulation of glycolysis via the AKT/mTOR/HIF1a pathway may be an important mechanism of chemoresistance in ALL and that mTOR inhibition or glucose normalization may play an important role in chemosensitization and improved outcomes in ALL.