1. University of Pennsylvania School of Medicine Department of Radiology
Metabolic flux analysis of mantle cell lymphoma cells upon Bruton tyrosine kinase inhibition
Seung-Cheol Lee, PhD
University of Pennsylvania School of Medicine
Department of Radiology

2. 13C 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

3. Melanoma cells, Dynamic MFA
Shestov, Mancuso and Lee et al., Journal of Biological Chemistry 2016

4. 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

5. Differential response of MCL cells to BTK inhibitor
Jeko RL

6. 1H NMR 48 hr treatment with 500 nM ibrutnib p=0.0003 p=0.01 p=0.003

7. Metabolite 13C enrichment determination (Proton Observe Carbon Edited NMR)
2.4
2.2
2.0
1.8
1.6
1.4
1.2
PPM
2.6
12C Glu
12C Ala
12C Lac
13C Glu
13C Ala
13C Lac
RL cells
Jeko cells
p=0.01
p=0.02
48 hr treatment + incubation with [1,6]glucose for 8 hr

8. Glutamate mulitiplet ratio determination
13C NMR
34.8
34.6
34.4
34.2
34.0
33.8
PPM 4s
4d34
28.2
28.0
27.8
27.6 3s
3d43
Glu4
Glu3
RL cells
Jeko cells
p=0.03

9. Mass balance equations
Chemical mass balance
13C isotope balance

10. Metabolic network

11. Determined metabolic fluxes

12. Correlation with RNA-seq analysis
* data not shown due to very low gene expression of this LDH isoform in JeKo-1 cells

13. Treatment of MCL cells with a glutaminase inhibitor
Cell growth (MTT assay)

14. Summary
We have determined various metabolic fluxes of MCL cells (RL and Jeko) upon perturbation with BTK inhibition by using 13C 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.

15. Acknowledgement
NIH R01-CA172820
R01-CA129544