Metabolic flux analysis of mantle cell lymphoma cells
Metabolic flux analysis of mantle cell lymphoma cells upon Bruton tyrosine kinase inhibition Seung-Cheol Lee, Ph. D University of Pennsylvania School of Medicine Department of Radiology
13 C Metabolic Flux Analysis (MFA) • Chance (1984) – heart, NMR • Malloy (1988) – heart, NMR • Mason, Gruetter, Rothman (1995) – brain, NMR • Wiechert (1997) – microorganism, NMR, MS • Stephanopoulos (2009) – cancer cells, MS • Shestov (2016) – cancer cells, NMR
Melanoma cells, Dynamic MFA Shestov, Mancuso and Lee et al. , Journal of Biological Chemistry 2016
Application of MFA to lymphoma cells and BTK inhibitor • • Bruton tyrosine kinase (BTK) inhibitor Mantle cell lymphoma (MCL) Cytostatic drug Find which pathways are affected by the inhibitor and determine metabolic pathways to be further targeted
Differential response of MCL cells to BTK inhibitor Jeko RL
1 H NMR p=0. 0003 mole/cell 5 E-14 6 E-14 RL cells cont ibr 4 E-14 3 E-14 p=0. 01 2 E-14 p=0. 0006 3 E-14 2 E-14 1 E-14 0 0 Ala Glu PCho 48 hr treatment with 500 n. M ibrutnib cont ibr 4 E-14 1 E-14 Lac Jeko cells 5 E-14 mole/cell 6 E-14 p=0. 003 p=0. 006 Lac Ala Glu PCho
Metabolite 13 C enrichment determination (Proton Observe Carbon Edited NMR) 12 C Lac p=0. 01 p=0. 02 Enrichment 12 C Ala RL cells cont ibr 0. 8 12 C Glu 0. 6 0. 4 0. 2 0 2. 4 2. 2 2. 0 1. 8 1. 6 1. 4 Lac 1. 2 PPM 13 C Lac Ala cont ibr 0. 7 13 C Glu 13 C Ala Glu 4 Glu 3 Glu 2 0. 9 Enrichment 48 hr treatment + incubation with [1, 6]glucose for 8 hr 1 0. 5 Jeko cells 0. 3 0. 1 2. 6 2. 4 2. 2 2. 0 1. 8 1. 6 1. 4 1. 2 PPM -0. 1 Lac Ala Glu 4 Glu 3 Glu 2
Glutamate mulitiplet ratio determination 13 C NMR 1 0. 8 0. 6 4 s 0. 4 Glu 4 4 d 34 34. 8 34. 6 34. 4 34. 2 34. 0 33. 8 PPM 3 d 43 3 s Glu 3 p=0. 03 RL cells cont ibr 0. 2 0 Glu 4 D/S Glu 3 D/S 3 d 43 28. 2 28. 0 27. 8 27. 6 PPM 1 0. 8 0. 6 0. 4 0. 2 Jeko cells cont ibr 0 Glu 4 D/S Glu 3 D/S
Mass balance equations • Chemical mass balance • 13 C isotope balance
Metabolic network
Determined metabolic fluxes
Correlation with RNA-seq analysis * data not shown due to very low gene expression of this LDH isoform in Je. Ko-1 cells
Cell growth (MTT assay) Treatment of MCL cells with a glutaminase inhibitor
Summary • We have determined various metabolic fluxes of MCL cells (RL and Jeko) upon perturbation with BTK inhibition by using 13 C NMR metabolic flux analysis. • Glutaminolytic pathway was found to be unaffected by BTK inhibition in Jeko cells. • Treatment of Jeko cells with a glutaminase inhibitor resulted in profound growth inhibition.
Acknowledgement • NIH R 01 -CA 172820 R 01 -CA 129544
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