Abstract 4328: Marrow stromal cells promote mitochondrial energy metabolism in primary CLL cells without impacting cellular proliferation
Authors: Vangapandu Hima Venkata , Balakrishnan Kumudha , Wierda William G. , Keating Michael J. , Stellrecht Christine M. , Gandhi Varsha 
Published: May 2015

Abstract
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of replicationally quiescent mature B cells, as they fail to undergo apoptosis. Studies show that supporting stromal cells in the microenvironment provide survival advantage to CLL cells by conferring resistance to spontaneous and drug-induced apoptosis. A recent transcriptome characterization showed that compared to normal lymphocytes, genes involved in metabolic pathways were upregulated in CLL cells (Ferreira et al., Genome Res 2013). We hypothesized that stromal cells may have an active role on energy metabolism of malignant B-cells in order to cope with their energy demands. We assessed the major energy generating pathways (glycolysis and mitochondrial OXPHOS), in primary CLL cells using Extracellular Flux Analyzer (Seahorse Bioscience). The oxygen consumption rate (OCR) in CLL cells is surprisingly high compared to the proliferating lymphoma cell lines. Whereas, the glycolytic flux, measured as extracellular acidification rate (ECAR) is significantly low, suggesting that OXPHOS measured as OCR is upregulated in CLL cells; hence we focused on OXPHOS. Increased OCR was not impacted by age, Rai stage, lymphocyte counts, β2M, IgVH mutation status or LDH levels. However, ZAP70 positive (a poor-prognosis marker) samples had higher OCRs (n=30, p =0.002). Compared to males, samples obtained from female patients had higher OCRs (n=33, p = 0.043). Co-culturing with stroma (NK.tert human cell line) for 18-24 hrs had an impact on OCR in CLL; out of 30 samples, 24 showed a significant (p - 0.0005) increase in basal OCR and/or spare respiratory capacity (SRC, biological parameter of mitochondrial respiration). In addition, 11 of 12 CLL samples showed a significant increase in basal OCR, when plated on stroma without prior incubation (p &lt; 0.0001), whereas the SRC did not statistically change (p - 0.9791). Moreover, the differences associated with gender and ZAP70 status were more pronounced when CLL cells were co-cultured on stroma (ZAP70 status, n= 30, p = 0.0013; Gender n=28, p =0.0173). In contrast to OCR, ECAR did not show stroma-induced statistical increase. Preliminary data with murine stroma line (M2-10B4) also showed upregulation of OCR in CLL cells. The stroma-mediated increase in OCR was not due to an increase in proliferation index; as CLL cells on stroma stained negative for Ki67 (marker for cell proliferation). Metabolite analysis by mass spectrometry of 5 CLL sample sets revealed that TCA cycle and gluconeogenesis were among the top 5 hits of pathways upregulated by stromal co-culture. At the molecular level, CLL cells in presence of stroma showed an increased p-AKT and p-ERK levels. Active AKT modulates GLUT4 protein levels, which was expressed in all 5 samples. Collectively, these data suggest that in CLL cells, OXPHOS (measured as OCR) is high and was further induced by stroma.
Citation Format: Hima Venkata Vangapandu, Kumudha Balakrishnan, William G. Wierda, Michael J. Keating, Christine M. Stellrecht, Varsha Gandhi. Marrow stromal cells promote mitochondrial energy metabolism in primary CLL cells without impacting cellular proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4328. doi:10.1158/1538-7445.AM2014-4328