Sullivan Lab Poster 2021 HBretreat Sep18 vF
1
Reductive Pyruvate Carboxylation Supports Aspartate Biosynthesis in SDH-deficient Cancer Cells Madeleine L. Hart 1,2 and Lucas B. Sullivan 1 1. Human Biology Division, Fred Hutch Cancer Research Center, Seattle, WA 98109 2. Department of Pathology, University of Washington, Seattle, WA 98106 A primary role of the electron transport chain is to support aspartate biosynthesis through NAD+ regeneration Succinate Dehydrogenase (SDH) is a tumor suppressor, but is also required for canonical aspartate synthesis How is SDH loss metabolically tolerated in proliferating cells? Aspartate Biomass SDH TCA cycle Succinate Dehydrogenase 1. SDH inhibition blocks proliferation, which is restored by aspartate but not electron acceptors 0.25 0.5 1 2 4 Relative Ratio (log2) NAD+/NADH PYR: - + - + - + Vehicle Rotenone Atpenin -0.5 0.0 0.5 1.0 1.5 Proliferation Rate (doublings / day) Vehicle Rotenone Atpenin PYR AKB NT ASP 2. SDH deficient cancer cells require complex I inhibition for proliferation, reduced redox, and aspartate synthesis 4. Complex I inhibition enables reductive pyruvate carboxylation to support ASP biosynthesis in SDH deficient cells Results Introduction 3. Mitochondrial transhydrogenase activity is required to support alternative aspartate biosynthesis Conclusions & Future Directions Acknowledgments Funding: T32 NIH GM095421; R00CA218679 NCI DS A big thank you to Lucas, the Sullivan Lab, Brian from Proteomics, the M3D PhD program at UW, Fred Hutch, and my supervisory committee. 0.0 0.5 1.0 1.5 Proliferation Rate (doublings / day) Atpenin (5 μM): Rotenone (50 nM): ✱✱✱ - - + - + + ns A B C D F G Figure 1. A) Both rotenone (CI) and atpenin (SDH) impair cell proliferation in the No Treatment condition (NT). Proliferation defects from rotenone treatment are completely overcome with the addition of electron acceptors (PYR, AKB). Proliferation is only partially restored by electron acceptors upon SDH inhibition with atpenin. Exogenous aspartate (ASP) restores proliferation of both rotenone and atpenin treated cells equally. B) PYR restores ASP levels in rotenone treated cells, but not atpenin treated cells. C) Pyruvate supplementation restores a decreased NAD+/NADH ratio in rotenone treated cells, while atpenin increases it. Figure 2. A) Complex I inhibition (rotenone) rescues the lowered proliferation rates caused by SDH inhibition (atpenin). B) Complex I inhibition lowers the high NAD+/NADH ratio from SDH inhibition. C) Complex I inhibition increases aspartate levels in SDH impaired cells. D) Complex I inhibition is required for single cell colony formation in SDHB KO cells. E) Proliferation of SDHB KO cells are restored by complex I inhibitors (rotenone & metformin) and exogenous aspartate. F) Re-expression of SDHB in SDHB KO cells restores proliferation and brings back atpenin sensitivity and rotenone rescue of SDH-impairment. G) Proliferation of SDHB null renal cell carcinoma cells (UOK269) is exacerbated by inducible expression of yeast complex I (NDI1) but are unaffected when SDHB is added back to these cells. H) Aspartate levels are decreased in SDHB-null UOK269s upon expression of yeast complex I (NDI1). § SDH deficient cells are limited for aspartate, which can be overcome by complex I inhibition § Complex I loss reprograms metabolism by altering redox status and utilizing mitochondrial transhydrogenase pathways to power non-canonical aspartate synthesis § In this model, newly synthesized aspartate is preferentially derived from glucose, requires mitochondrial pyruvate entry, and is terminally converted to aspartate in the cytosol § Complete mechanism through KOs, tracing, and proliferation assays § In vivo studies § Assess adaptation to lose complex I over time in SDHB null cancer cells Figure 3. A) NADPH depletion in the cytoplasm through expression of cytoTPNOX has no effect on rotenone rescue of SDH impaired cells. B) Orthogonal mitochondrial NADH oxidation with mitoLbNOX expression or C) blocking mitochondrial NADP(H) production by NADK2 KO abolish proliferation rescue from rotenone. A H E 0.0 0.5 1.0 1.5 Proliferation Rate (doublings / day) Atpenin (5 μM): Rotenone (50 nM): - - + - + + mitoLbNOX ns Sullivan et al. 2015, Cell Birosy et al. 2015, Cell 0.0 0.5 1.0 1.5 SDHB AddBack Proliferation Rate (doublings / day) Rotenone (50 nM): Atpenin (5 μM): - - + - - + + + B C 0.0 0.5 1.0 Proliferation Rate (normalized d -1 ) - PC MPC1 GOT1 GOT2 KO: C D E F @madelouhart Figure 4. A) Mass incorporation (M+X) identifies aspartate production pathway from glutamine. B) Rotenone treatment affects aspartate levels derived from 13 C glutamine (height) and carbon source (colored segments) in WT and SDHB KO cells. C) 13 C glucose produces M+3 aspartate by pyruvate carboxylation. D) Rotenone treatment affects aspartate levels (height) and carbon source (colored segments) from 13 C glucose in WT and SDHB KO cells. E) Proliferation rate of cell lines with genetic knockout (KO) of metabolic genes with ETC inhibitors, normalized for vehicle proliferation rates F) suggest a novel path of aspartate synthesis.
Transcript of Sullivan Lab Poster 2021 HBretreat Sep18 vF
Reductive Pyruvate Carboxylation Supports Aspartate Biosynthesis in SDH-deficient Cancer Cells
Madeleine L. Hart1,2 and Lucas B. Sullivan1
1. Human Biology Division, Fred Hutch Cancer Research Center, Seattle, WA 981092. Department of Pathology, University of Washington, Seattle, WA 98106
A primary role of the electron transport chain is to support aspartate biosynthesis through NAD+ regeneration
Succinate Dehydrogenase (SDH) is a tumor suppressor, but is also required for canonical aspartate synthesis
How is SDH loss metabolically tolerated in proliferating cells?
AspartateBiomass
SDH
TCA cycleSuccinate Dehydrogenase
1. SDH inhibition blocks proliferation, which is restored by aspartate but not electron acceptors
0.25
0.5
1
2
4
Rel
ativ
e R
atio
(log
2)
NAD+/NADH
PYR: - + - + - + Vehicle Rotenone Atpenin
-0.5
0.0
0.5
1.0
1.5
Pro
lifer
atio
n R
ate
(do
ub
ling
s / d
ay) Vehicle
Rotenone
Atpenin
PYR AKBNT ASP
2. SDH deficient cancer cells require complex I inhibition for proliferation, reduced redox, and aspartate synthesis
4. Complex I inhibition enables reductive pyruvate carboxylation to support ASP biosynthesis in SDH deficient cells
Results
Introduction
3. Mitochondrial transhydrogenase activity is required to support alternative aspartate biosynthesis
Conclusions & Future Directions
Acknowledgments
Funding: T32 NIH GM095421; R00CA218679 NCI DS
A big thank you to Lucas, the Sullivan Lab, Brian from Proteomics, the M3D PhD program at UW, Fred Hutch, and my supervisory committee.
0.0
0.5
1.0
1.5
Prol
ifera
tion
Rat
e (d
oubl
ings
/ da
y)
Atpenin (5 µM):Rotenone (50 nM):
✱✱✱
--
+-
++
nsA B C
D F
G
Figure 1. A) Both rotenone (CI) and atpenin (SDH) impair cell proliferation in the No Treatment condition (NT). Proliferation defects from rotenone treatment are completely overcome with the addition of electron acceptors (PYR, AKB). Proliferation is only partially restored by electron acceptors upon SDH inhibition with atpenin. Exogenous aspartate (ASP) restores proliferation of both rotenone and atpenin treated cells equally. B) PYR restores ASP levels in rotenone treated cells, but not atpenin treated cells.C) Pyruvate supplementation restores a decreased NAD+/NADH ratio in rotenone treated cells, while atpenin increases it.
Figure 2. A) Complex I inhibition (rotenone) rescues the lowered proliferation rates caused by SDH inhibition (atpenin). B) Complex I inhibition lowers the high NAD+/NADH ratio from SDH inhibition. C) Complex I inhibition increases aspartate levels in SDH impaired cells. D) Complex I inhibition is required for single cell colony formation in SDHB KO cells. E) Proliferation of SDHB KO cells are restored by complex I inhibitors (rotenone & metformin) and exogenous aspartate. F) Re-expression of SDHB in SDHB KO cells restores proliferation and brings back atpenin sensitivity and rotenone rescue of SDH-impairment. G) Proliferation of SDHB null renal cell carcinoma cells (UOK269) is exacerbated by inducible expression of yeast complex I (NDI1) but are unaffected when SDHB is added back to these cells. H) Aspartate levels are decreased in SDHB-null UOK269s upon expression of yeast complex I (NDI1).
§ SDH deficient cells are limited for aspartate, which can be overcome by complex I inhibition
§ Complex I loss reprograms metabolism by altering redox status and utilizing mitochondrial transhydrogenase pathways to power non-canonical aspartate synthesis
§ In this model, newly synthesized aspartate is preferentially derived from glucose, requires mitochondrial pyruvate entry, and is terminally converted to aspartate in the cytosol
§ Complete mechanism through KOs, tracing, and proliferation assays
§ In vivo studies
§ Assess adaptation to lose complex I over time in SDHB null cancer cells
Figure 3. A) NADPH depletion in the cytoplasm through expression of cytoTPNOX has no effect on rotenone rescue of SDH impaired cells. B) Orthogonal mitochondrial NADH oxidation with mitoLbNOX expression or C) blocking mitochondrial NADP(H) production by NADK2 KO abolish proliferation rescue from rotenone.
A
H
E
0.0
0.5
1.0
1.5
Prol
ifera
tion
Rat
e (d
oubl
ings
/ da
y)
Atpenin (5 µM):Rotenone (50 nM):
--
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++
mitoLbNOX
ns
Sullivan et al. 2015, CellBirosy et al. 2015, Cell 0.0
0.5
1.0
1.5 SDHB AddBack
Prol
ifera
tion
Rat
e (d
oubl
ings
/ da
y)
Rotenone (50 nM):Atpenin (5 µM):
--
+-
-+
++
B C
0.0
0.5
1.0
Prol
ifera
tion
Rat
e (n
orm
aliz
ed d
-1)
- PC MPC1 GOT1 GOT2KO:
C D
E F
@madelouhart
Figure 4. A) Mass incorporation (M+X) identifies aspartate production pathway from glutamine. B) Rotenone treatment affects aspartate levels derived from 13C glutamine (height) and carbon source (colored segments) in WT and SDHB KO cells. C) 13C glucose produces M+3 aspartate by pyruvate carboxylation. D) Rotenone treatment affects aspartate levels (height) and carbon source (colored segments) from 13C glucose in WT and SDHB KO cells. E) Proliferation rate of cell lines with genetic knockout (KO) of metabolic genes with ETC inhibitors, normalized for vehicle proliferation rates F) suggest a novel path of aspartate synthesis.
