Abstract 2658: Stromal microenvironment modulates mitochondrial metabolism in chronic lymphocytic leukemia cells and is abrogated by PI3K δ and γ inhibition
Authors: Vangapandu Hima V. , Balakrishnan Kumudha , Ayres Mary L. , Wierda William G. , Keating Michael J. , Stellrecht Christine M. , Gandhi Varsha 
Published: August 2015

Abstract
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of replicationally quiescent mature B cells, as they fail to undergo apoptosis. Supporting stromal cells in the microenvironment provide survival advantage to CLL cells. We hypothesized that stromal cells may have an active role in energy metabolism of malignant B-cells in order to cope with their energy demands and is driven by PI3K pathway. We assessed the two major energy generating pathways (glycolysis and mitochondrial Oxidative Phosphorylation (OXPHOS)), in primary CLL cells using Extracellular Flux Analyzer (Seahorse Bioscience), in presence of three different stromal cell lines. Glycolysis measured as ECAR did not show stroma-induced increase, which is commonly upregulated in most cancers. A decrease in glucose uptake in coculture experiments, further suggested that glycolysis was not upregulated in CLL cells (n = 7, p&lt;0.01). Surprisingly, OXPHOS measured as OCR in CLL cells from 29 patients showed a statistically significant increase, (p&lt;0.01) upon coculture and contact with NK-Tert stromal cells. Similar OCR induction was observed in presence of M2-10B4 and HS-5 stromal cell lines. However, no significant changes were observed in mitochondrial mass, membrane potential and ROS, upon co-culture. All four ribonucleotide triphosphate pools (ATP, CTP, GTP, and UTP), showed a 2-fold and statistically significant increase after interacting with stroma cell line for 48hr. Changes in metabolomics and NTP pools were not due to cell growth as stroma cocultures did not impact proliferation index (Ki-67 staining) of CLL cells. In parallel to these changes in metabolomics, stromal microenvironment increased AKT phosphorylation at Thr308 and Ser473 indicating upregulation of PI3K pathway. Consistently, inhibition of PI3K axis by IPI-145 (duvelisib), a PI3K δ and γ inhibitor, currently in phase III trials for CLL, decreased phospho-AKT in CLL samples (n = 6) and dramatically reduced OCR (n = 7) and ECAR in CLL cells in suspension cultures without causing cytotoxicity. Importantly, stroma-mediated increase in OCR in CLL cells was significantly compromised by IPI-145 treatment (n = 5; p&lt;0.05). In parallel, stroma-driven AKT phosphorylation in CLL samples (n = 6) was also reduced with IPI-145. Preliminary data also indicates a decline in NTP pools, upon drug treatment. Collectively, these data suggest that stroma impacts metabolomics in quiescent CLL cells which is in part driven by PI3K/AKT pathway.
Citation Format: Hima V. Vangapandu, Kumudha Balakrishnan, Mary L. Ayres, William G. Wierda, Michael J. Keating, Christine M. Stellrecht, Varsha Gandhi. Stromal microenvironment modulates mitochondrial metabolism in chronic lymphocytic leukemia cells and is abrogated by PI3K δ and γ inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2658. doi:10.1158/1538-7445.AM2015-2658