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Review

Mitochondria and Hypoxia: Metabolic


Crosstalk in Cell-Fate Decisions
David Bargiela,1,2 Stephen P. Burr,1,2 and Patrick F. Chinnery1,2,*

Alterations in mitochondrial metabolism influence cell differentiation and Highlights


growth. This process is regulated by the activity of 2-oxoglutarate (2OG)- Alignment of transcriptional activity
dependent dioxygenases (2OGDDs) – a diverse superfamily of oxygen-con- with nutrient availability is crucial to
maintain homeostasis in mammalian
suming enzymes – through modulation of the epigenetic landscape and tran- cells. The 2-oxoglutarate-dependent
scriptional responses. Recent reports have described the role of mitochondrial dioxygenase (2OGDD) superfamily
integrates mitochondrial metabolic
metabolites in directing 2OGDD-driven cell-fate switches in stem cells (SCs), signals to coordinate downstream
immune cells, and cancer cells. An understanding of the metabolic mecha- transcriptional responses.
nisms underlying 2OGDD autoregulation is required for therapeutic targeting of
Oxygen and mitochondrial metabolite
this system. We propose a model dependent on oxygen and metabolite avail- concentrations signal via 2OGDDs to
ability and discuss how this integrates 2OGDD metabolic signalling, the hyp- alter DNA/histone methylation and
hypoxic transcriptional activity.
oxic transcriptional response, and fate-determining epigenetic changes.
Targeting 2OGDD activity affects cell
fate in vivo and has relevance in the
Introduction treatment of immune diseases and
Oxygen is essential for almost all animal life on the planet, allowing aerobic metabolism and the cancer.

efficient production of ATP via oxidative phosphorylation (OXPHOS). Consequently, multicel-


lular organisms have evolved a number of mechanisms to rapidly adapt to decreased oxygen
availability (termed hypoxia), prolonging survival in the absence of this critical resource.

Mitochondria serve as metabolic hubs in the cell and are intimately linked with the adaptive
response to hypoxia. A mitochondrial–nuclear link via 2OGDD enzymes allows crosstalk
between mitochondrial metabolism and nuclear transcriptional activity. The 2OGDD enzyme
family includes regulators of hypoxia-inducible factor (HIF), the master transcription factor
orchestrating cellular adaptation to hypoxia, as well as DNA and histone demethylase enzymes
involved in modifying the epigenetic landscape [1]. Recent work has shown that modulation of
2OGDD activity through these pathways is able to shape cell fate, with particular relevance to
immunity and cancer.

In this review we describe the key interactions between mitochondria and the hypoxia
response, focussing on the regulation of transcription by mitochondrial metabolism and the
2OGDD family. We offer a model of metabolic cross-regulation of 2OGDD family members,
1
MRC Mitochondrial Biology Unit,
illustrating cellular mechanisms that may represent promising future therapeutic targets.
Cambridge Biomedical Campus,
Cambridge CB2 0XY, UK
2
Hypoxia, HIFs, and the 2OGDDs Department of Clinical
Neurosciences, Cambridge
Several cellular mechanisms are involved in the adaptation to acute hypoxia, such as activation of ion
Biomedical Campus, University of
channels via gaseous signalling in carotid body glomus cells [2] and the direct upregulation of Cambridge, Cambridge CB2 0QQ, UK
glycolysis by AMPK in cardiac myocytes [3]. However, the major pathway controlling the cellular
response to hypoxia occurs at the transcriptional level and is coordinated through a number of key
*Correspondence:
transcriptions factors, resulting in upregulation of genes involved in metabolism, angiogenesis and,
pfc25@medschl.cam.ac.uk
haematopoiesis [4–6]. To date, the best studied of these transcriptional regulators are the HIFs. (P.F. Chinnery).

Trends in Endocrinology & Metabolism, April 2018, Vol. 29, No. 4 https://doi.org/10.1016/j.tem.2018.02.002 249
© 2018 Elsevier Ltd. All rights reserved.
HIFs are members of the basic helix–loop–helix (bHLH)/Per–Arnt–Sim (PAS) family of transcription
factors and form functional heterodimers comprising one of three known HIFa subunits and the
HIFb/aryl hydrocarbon receptor nuclear translocator (ARNT) subunit [6]. Active HIF heterodimers
formed in the nucleus bind DNA at conserved HIF-responsive elements (HREs) located in the
enhancer and promoter regions of over 200 HIF-target genes [7,8], including erythropoietin (EPO)
[9], vascular endothelial growth factor (VEGF) [10], and glucose transporter 1 (GLUT-1) [11],
enhancing the transcription of these genes and enabling tissues to adapt to hypoxia.

Wang and Semenza first identified that oxygen-dependent control of this transcriptional
response is conferred through the labile HIFa subunits (the major mammalian isoforms being
HIF1a and HIF2a/EPAS), which are stabilised in hypoxia but rapidly turned over under aerobic
conditions [9,12]. The key oxygen-sensing component of this pathway involves post-transla-
tional hydroxylation of two conserved proline residues of the HIFa protein by a family of prolyl
hydroxylase domain-containing proteins (PHDs) [13,14]. Following hydroxylation, the von
Hippel–Lindau (VHL) E3 ligase complex polyubiquitinates both HIF1a and HIF2a in an oxy-
gen-dependent manner [15,16]. This facilitates rapid proteasomal degradation of the HIFa
subunits in normoxia but is abolished on exposure to hypoxia, leading to stabilisation and
activation of HIF signalling [16,17] (Figure 1A).

PHDs are a critical component of the canonical hypoxia response pathway and also form part of a
larger group of 2OGDDs (Box 1). This enzyme family includes the asparaginyl hydroxylase factor
inhibiting HIF (FIH), a regulator of HIF transcriptional activity [18], as well as epigenetic modulators –
the Jumonji C-domain-containing histone demethylases (JHDs) [19,20] and the ten–eleven
translocation (TET) dioxygenase enzymes [21–23], which promote DNA demethylation. As their
name suggests, all 2OGDD members have a functional requirement for 2OG and oxygen, and
additionally utilise ferrous iron and ascorbate as cofactors [24] (Figure 1B, inset). Therefore,
changes in cofactor levels, such as reduced oxygen availability in hypoxic environments, result
in altered activity of these enzymes. Furthermore, competition for a finite cofactor pool may also
represent a level of regulation, integrating information on oxygen, metabolite, and macromolecule
availability with epigenomic and transcriptomic programmes, as discussed below.

Mitochondria and Hypoxia: Crosstalk via 2OGDDs


Mitochondria are metabolic hubs in cells and generate energy in the form of ATP by OXPHOS
[38]. As a major consumer of oxygen, mitochondrial OXPHOS has a significant impact on the
availability of molecular oxygen to regulate the hypoxic response and other 2OGDD-dependent
reactions. Multiple studies have shown that reduced activity of mitochondrial complex I,
through either chemical inhibition or genetic mutation, decreases mitochondrial oxygen con-
sumption, increasing oxygen availability in the cytoplasm, and destabilises both HIF1a and
HIF2a under hypoxic conditions [39–42]. There is evidence that cells may be able to actively
redistribute oxygen under hypoxic conditions and modulate HIF signalling through production
of endogenous ETC inhibitors, such as nitric oxide [43], or altered expression of proteins that
regulate mitochondrial oxygen consumption [44] (Figure 1A). Furthermore, HIF-mediated
transcription of PDK1 and LDHA is able to limit the entry of acetyl-CoA into the oxidative
tricarboxylic acid (TCA) cycle (by blocking the conversion of pyruvate to acetyl-CoA via PDK1
inhibition of PDH) and instead switches cells towards glycolytic metabolism [45–48].

A number of HIF-dependent mechanisms in turn also feed back on mitochondrial function,


highlighting the reciprocal relationship between these two key cellular processes. HIFs have
been shown to directly regulate the expression of a number of ETC subunits [49,50] and the
HIF-responsive noncoding miRNA-210 has been implicated in suppression of the iron–sulphur

250 Trends in Endocrinology & Metabolism, April 2018, Vol. 29, No. 4
(A) Normoxia Hypoxia

HydroxylaƟon
HIF1α
HIF1α
DimerisaƟon
UbiquiƟnaƟon OH
HIF1α
PHD
HIF1α HIF1β
Poly-Ub OH
HIF1α
VHL
HIF1β TranscripƟon
DegradaƟon
HRE

Nucleus
Proteasome

(B) 2OGDDs
O2 + + CO2
O2
O2 s
ink TETs EpigeneƟc
2-OG + Fe(II)
+ Ascorbate
Succinate

2OG remodelling PHDs


TCA cycle
JHDs HIF1α
ETC HIF1β TranscripƟon
HIF O O
ROS HRE
signaling
Mitochondrion Succinate PHDs HIF
N N HIF–OH
Fumarate Nucleus (stable)
(degraded)
2HG
ETC OH

components

Figure 1. 2-Oxoglutarate (2OG)-Dependent Dioxygenases (2OGDDs) Link Mitochondria and Transcriptional Control. Hypoxia-inducible factor 1 alpha
(HIF1a) is stabilised in hypoxic conditions, dimerises with HIF1 beta (HIF1b)/aryl hydrocarbon receptor nuclear translocator (ARNT), and binds to hypoxia response
elements (HREs) to enhance transcription at target genes (A, right). Under normoxic conditions (A, left), HIF1a undergoes prolyl hydroxylase domain-containing protein
(PHD)-mediated hydroxylation and polyubiquitinylation by the von Hippel–Lindau (VHL) E3 ligase complex followed by proteasomal degradation. 2OGDD’s regulation of
transcriptional activity is dependent on oxygen and 2OG and is inhibited by mitochondrial metabolites [succinate, fumarate, 2-hydroxyglutarate (2HG)] and reactive
oxygen species (ROS) (B). 2OGDDs convert 2OG and oxygen (O2) to succinate and carbon dioxide (CO2), respectively (B, inset). Ferrous iron [Fe (II)] and ascorbate serve
as further cofactors. Abbreviations: ETC, electron transport chain; JHDs, Jumonji domain-containing histone demethylase enzymes; Me, methyl; TCA, tricarboxylic
acid; TETs, ten–eleven translocation dioxygenase enzymes.

cluster assembly proteins ISCU1 and 2, which are critical for the function of a number of ETC
components [51,52]. These feedback loops between mitochondria and the HIFs are likely to be
involved in the fine-tuning of both metabolic and transcriptional responses to hypoxia.

Reactive oxygen species (ROS) from the mitochondrial ETC (mROS) were originally thought to
be merely a harmful byproduct of OXPHOS, but it is now clear that they function as signalling
molecules in a wide range of cellular processes. The production of mROS during hypoxia may
represent an important mito-hypoxia signalling mechanism. Chandel et al. first reported that
mROS produced by mitochondrial complex III during hypoxia are able to inhibit PHDs and
stabilise HIFa, suggesting a role for mROS in the activation of HIF under hypoxic conditions
[53]. Later studies supported this mechanism, showing that blockade of mROS production,
either genetically or pharmacologically, resulted in impaired induction of HIF in hypoxia [54–56].
mROS-mediated inhibition of HIF has been proposed to occur via oxidation of the cysteine
residues or Fe(II) cofactor [57] required for PHD2 activity [57–59] or via inhibition of FIH [60].

Trends in Endocrinology & Metabolism, April 2018, Vol. 29, No. 4 251
Box 1. The 2OGDDs
The 2OGDDs are a family of over 60 enzymes responsible for catalysing a number of key oxidative modifications in a
diverse range of target proteins [25]. The active site of the 2OGDDs comprises a characteristic ‘jelly-roll’ motif
comprising two b sheets within which an Fe(II) atom is bound by conserved histidine and aspartate residues [26].
The iron-bound holoenzyme binds 2-OG to form a stable enzyme–Fe(II)–2-OG complex [27], which then associates with
the target protein. In the presence of molecular oxygen, 2-OG is decarboxylated, resulting in the release of CO2,
production of succinate, and oxidation of the target [24]:
RH þ O2 þ C5 H6 O5 ! ROH þ CO2 þ C4 H6 O4 : [I]

In addition to Fe(II) and 2-OG, many of the 2OGDDs also utilise ascorbate as a cofactor, although this may not be
absolutely required [25].

The 2OGDDs catalyse a range of important post-translational protein modifications [28] and this family of enzymes is
responsible for regulating a number of key intracellular pathways in both prokaryotic and eukaryotic cells [29]. Perhaps
the best-studied of the 2OGDD family members in recent years include the HIF PHDs responsible for oxygen sensing
and control of the hypoxic response [30,31], the TET dioxygenases, which hydroxylate 5-methylcytosine and promote
DNA demethylation [32,33], and the Jumonji-C demethylases, which play a crucial role in histone demethylation [34,35].

Since these enzymes require both oxygen and the mitochondrial intermediate metabolite 2OG to function, their activity
can be significantly influenced by the metabolic state of the cell, providing a key link between metabolism and major
intracellular signalling pathways such as the hypoxic response and epigenetic regulation [36,37].

The regulation of OXPHOS by metabolic intermediates and oxygen allows mitochondria to


respond to alterations in nutrient availability, re-establishing homeostasis by redirecting metabolic
flux to compensate. This metabolic reprogramming may represent an important mechanism of
mitochondrial–nuclear signalling whereby the energy status of the cell is kept in sync with the
epigenetic and transcriptional responses required for cell proliferation and differentiation. These
responses in turn influence oxygen supply and usage, tightly regulating ROS, which, although
functioning as key signalling molecules, may also be toxic to the cell when in excess. The position of
2OG at the intersection of catabolic pathways facilitates its role as a rheostat of glucose and fatty
acid metabolism (via TCA cycle activity) as well as glutamine metabolism (by direct production via
glutamate). The availability and flux of 2OG is then able to regulate 2OGDD activity by altering the
ratio of promoting and inhibiting metabolites, discussed further in the following section.

The 2OGDD Shared Cofactor Pool (SCP) Model: Evidence and Implications
Metabolic regulation of transcriptional activity via 2OGDDs is involved in determining cell fate in
development and disease. In this section we briefly discuss another metabolic–transcriptional
process, the removal of epigenetic marks via 2OG-dependent demethylase enzymes, the
activity of which relates to the mitochondrial HIF response through a shared dependency on
2OGDD substrates and cofactors. By integrating these 2OGDD-dependent processes, we
propose a cross-regulation model that allows 2OGDD cofactor levels and activity to be
coordinated across multiple pathways (Figure 2).

As the epigenetic modulators of the 2OGDD family, the JHDs and TET demethylases (TETs) aid
the removal of methylation marks from histones and DNA, respectively, oxidising repressive
methyl groups to a hydroxymethyl form as an initial step in this process [20–22].

2OG serves as a co-substrate for this reaction and is converted to succinate and then fumarate.
Due to their structural similarity to 2OG, succinate and fumarate are able to inhibit enzyme
activity by competing for the 2OG-binding site [61], forming a regulatory loop (Figure 2B).

The same competitive inhibition occurs in the regulation of the hypoxia HIF transcriptional
response. PHDs and FIH require 2OG and other 2OGDD cofactors to regulate the abundance

252 Trends in Endocrinology & Metabolism, April 2018, Vol. 29, No. 4
(A) Mitochondrial metabolism
Mitochondrial metabolites
Forward Reverse Glucose
Glucose
(oxidaƟve) flux
Macromolecular synthesis (reducƟve) flux FaƩy acid
synthesis

Citrate Acetyl-CoA

TCA cycle TCA cycle


2HG
ETC ETC
2OG
Fumarate 2OG O2
ROS
Glutamine Glutamine
Succinate 2OGDD shared
co-factor pool
NucleoƟde/protein
synthesis
Fe2+
CompeƟƟve 2OG CompeƟƟve
inhibiƟon Ascorbate inhibiƟon
(B) EpigeneƟc remodelling (C) HIF acƟvity
O2 O2
2OG
CO2 Proteosomal
Succinate PHD OH
O2 degradaƟon
JHD 2OG
Succinate
hMe
Me CO2 HIF1α
Cytosol
TET/JHD PHD/FIH O2 Nucleus
2OG
acƟvity acƟvity HIF1α/β
FIH

5mC 5hmC
Succinate TranscripƟon
CO2 +
TET Succinate
2OG CompeƟƟve
O2 CO2 HRE
inhibiƟon

Figure 2. The 2-Oxoglutarate (2OG)-Dependent Dioxygenase (2OGDD) Shared Cofactor Pool Model. 2OG is oxidised to form succinate and then
fumarate or reduced to form citrate or 2-hydroxyglutarate (2HG) (A). The direction of tricarboxylic acid (TCA) flux depends on the redox environment and the availability of
downstream metabolites: an increased 2OG:succinate ratio promotes oxidative flux while an increased 2OG:citrate ratio promotes reductive flux. TCA activity relates to
oxidative phosphorylation (OXPHOS) activity via the redox state of electron carriers (e.g., NADH/FADH2) and oxygen availability in the mitochondria. 2OGDDs, involved
in epigenetic remodelling [mediated by the oxidase activity of ten–eleven translocation dioxygenase (TET)/Jumonji domain-containing histone demethylase (JHD)
enzymes] (B), and regulation of the hypoxia/hypoxia-inducible factor (HIF) response (C) both depend on 2OG and cofactor availability for their activity. 2OGDDs
competitively inhibit each other due to a limited supply of 2OGDD cofactors in the cell. Abbreviations: 5hmC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine; ETC,
electron transport chain; FIH, factor inhibiting HIF; hME, hydroxymethyl; HRE, hypoxia-response element; Me, methyl; PHD, prolyl hydroxylase enzymes; ROS, reactive
oxygen species.

and transcriptional activity of HIF, respectively. Competition for the 2OG-binding site by
succinate or fumarate inhibits the action of PHDs, causing stabilisation of HIF1 under normoxic
conditions, termed pseudohypoxia [62,63]. In tumours with mutations in mitochondrial succi-
nate dehydrogenase (SDH) or fumarate hydratase (FH), succinate or fumarate accumulate
resulting in the inhibition of PHDs and promotion of protumorigenic HIF transcription, including
VEGF-mediated angiogenesis and glycolytic metabolism [63,64]. Likewise, high levels of
succinate and fumarate inhibit the TET and JHD enzymes, leading to hypermethylation of
cell-differentiation genes and promoting malignant growth [65–67]. In both cases inhibition via
succinate or fumarate can be rescued by the addition of cell-permeable 2OG, restoring 2OGDD
activity by outcompeting the inhibitory metabolites [68,69].

Control of the intracellular 2OG:succinate ratio has also been shown to be crucial for the
maintenance of SC pluripotency. Initial studies in mouse embryonic SCs (mESCs) identified
elevated 2OG levels in naïve mESCs resulting in an elevated 2OG:succinate ratio and conse-
quent promotion of histone and DNA demethylation via increased TET/JHD activity [70].
Manipulation of the 2OG:succinate ratio directly impacts mESC cell-fate determination, with

Trends in Endocrinology & Metabolism, April 2018, Vol. 29, No. 4 253
the addition of cell-permeable 2OG or succinate being sufficient to promote mESC pluripotency
or differentiation, respectively [70,71]. The role of 2OG in maintaining stemness has also been
explored in naïve human pluripotent SCs (hPSCs) [72]. However, primed SCs from both mice
and humans have been shown to respond differently, with increased 2OG levels promoting
differentiation rather than maintaining pluripotency despite similar increases in TET and JHD
activity.

2OG can also undergo reductive carboxylation to isocitrate via the enzyme isocitrate dehy-
drogenase (IDH) (Figure 2A). Reductive flux is promoted by increases in the NADH:NAD+ and
2OG:citrate ratios; however, it is dependent on continued oxidation of 2OG to maintain
sufficient levels of NADPH [73]. Hypoxia also promotes reductive carboxylation of 2OG,
providing a crucial carbon source for fatty acid synthesis [74,75]. In cancer cells with mito-
chondrial respiratory chain complex defects, reductive metabolism of 2OG promotes prolifer-
ation [76], and this process may also influence tumour invasiveness by supporting anchorage-
independent growth [7].

Somatic gain-of-functions mutation in IDH, detected in haematological malignancies and solid


tumours, result in the conversion of 2OG to the oncometabolite R-2-hydroxyglutarate (R-2HG)
[77,78]. Structurally similar to 2OG, 2HG is a chiral molecule that exists as two enantiomers, S-
and R-2HG (also known as L- and D-2HG, respectively). Both are produced endogenously in
cells, as evidenced by the presence of specific dehydrogenase enzymes for both the S- and R-
forms [79,80]. However, the biochemical properties of the two enantiomers appear to differ
significantly. R-2HG, but not S-2HG, drives malignant transformation in the context of acute
myeloid leukaemia with IDH mutations [81] despite both 2-HG enantiomers being able to block
TET enzymes [82–84]. This differential response in promoting leukaemogenesis is attributed to
the ability of S-2HG, but not R-2HG, to inhibit PHD enzymes [81]. However, the role of R-2HG in
regulating 2-OGDDs under physiological conditions remains unclear.

S-2HG demonstrates potent inhibition of 2OGDDs both in vitro and in vivo. Metabolomic
profiling of cells in hypoxia revealed significant accumulation of S-2HG [85–87], with carbon-flux
analysis indicating that the source of S-2HG was 2OG derived from glutamine [86]. The
mechanism responsible for the conversion of 2OG to S-2HG appears to depend on the activity
of lactate dehydrogenase A (LDHA) and malate dehydrogenase (MDH) 1 and 2, with all three of
these enzymes potentially contributing to S-2HG production [85,86], although the choice of
enzyme may be cell-type specific [87]. This promiscuous substrate usage by LDHA and MDH1/
2 is favoured at low pH [88,89] and in the context of a low NAD+:NADH ratio [85,90], both of
which occur in hypoxia.

Elevated S-2HG levels have also been seen in normoxic conditions following disruption of the
oxoglutarate dehydrogenase complex (OGDHC) [91] and in fetal mouse haematopoietic SCs
with mitochondrial complex III deficiency [90], suggesting that metabolic defects can create a
permissive redox environment facilitating the accumulation of S-2HG. Tyrakis et al. recently
showed that the activation signal in T cells is sufficient to cause an increase in intracellular S-
2HG levels under normoxic conditions. This effect was amplified when cells were cultured in
hypoxia, resulting in inhibition of TET enzymes and the acquisition of a CD8+ memory-like
phenotype [87]. It is clear that a number of complex mechanisms regulate 2HG concentrations
and enantiomer ratios; further work is required to clarify how these processes may skew
immune responses in autoimmune disease or cancer.

254 Trends in Endocrinology & Metabolism, April 2018, Vol. 29, No. 4
Concluding Remarks and Therapeutic Prospects Outstanding Questions
The involvement of 2OGDDs in pathological processes such as cancer, inflammation, and How do primary mitochondrial disor-
anaemia have made them attractive drug targets. Pharmacological efforts to date have been ders alter 2OGDD’s regulation of cell
fate?
directed towards the dioxygenase enzymes themselves as well as the cofactors that regulate
their activity. How do the actions of the 2-HG enan-
tiomers R-2HG and S-2HG differ in
Small-molecule inhibitors of mutant IDH were developed with the aim of inhibiting 2HG- vivo and what therapeutic implications
does this have in autoimmune disease
mediated inhibition of TET/JHD activity. These compounds showed encouraging results in
and cancer?
vitro using patient-derived leukaemia cells and in vivo in a murine xenograft model, in both
cases reversing aberrant methylation to restore cell differentiation [81,92–94]. Clinical trials in How does 2OGDD-driven HIF metab-
patients with IDH1/2-mutant cancers demonstrated a profile similar to preclinical studies, olism feed back to regulate 2OGDD
showing clinical evidence of restoration of cell differentiation [95,96], and recently enasidenib activity?
(Idhifa, Celgene Corp.) has gained FDA approval as a treatment for patients with relapsed or
What are the molecular mechanisms
refractory AML with IDH2 mutations [97]. Similarly, a number of inhibitors of the PHD enzymes
coordinating 2OGDD–HIF–TET/JHDM
are in Phase II/III clinical trials for the treatment of renal anaemia. These compounds target activity with other metabolic–transcrip-
PHD2 and stimulate endogenous production of EPO via HIF2a-mediated transcription to tional axes (e.g., acetyl-CoA–DNA/his-
promote red blood cell production [98]. However, the activation of other aspects of HIF tone acetylation) in the cell?

transcription, such as angiogenesis and reduction in ROS production, may allow repurposing
of these drugs for other indications; for example, to limit damage from ischaemic injury [99–
102].

Altering oxygen and cofactor concentrations to modulate 2OGDD activity may represent an
alternative therapeutic strategy. Mootha and colleagues recently demonstrated that blockade
of VHL, a member of the hypoxia response pathway downstream of 2OGDDs, may confer
survival benefits in cells with mitochondrial respiratory chain inhibition [103]. They were able to
replicate this therapeutic response in vivo by exposing mice with mitochondrial respiratory
chain complex defects to low oxygen concentrations that limited VHL activity. The use of
moderate hypoxia to treat primary and secondary mitochondrial diseases in humans is an
intriguing possibility, although questions regarding tolerability, off-target effects, and the
duration of hypoxic exposure required for therapeutic effect will need to be ascertained.

Ascorbic acid (AA) (also known as vitamin C), a 2OGDD cofactor, also modulates the hypoxia
response pathway by promoting PHD activity through reduction of the enzyme’s inactive Fe(III)
state to an active Fe(II) state, thus rescuing its catalytic potential [104]. It may act similarly in
promoting TET-dependent 5hmC formation [105–109], although whether it modulates TETs
directly or indirectly via nonspecific reduction of the cellular iron pool has been questioned
[110,111]. Due to the potential antitumorigenic effects of TET-mediated promotion of differen-
tiation or PHD-mediated restriction of the HIF translational response, there has been renewed
interest in the potential use of AA as a cancer treatment [112,113]. High-dose intravenous AA
therapy has been shown to inhibit tumor growth in rodent xenograft models [114–116] and was
well tolerated when administered to pancreatic cancer patients in two small clinical studies
[117,118]. A Phase II trial will assess the effects of AA as an adjunct to chemotherapy for
pancreatic cancer (ClinicalTrials.gov NCT02905578). Regarding hypoxic therapy, further work
will be required to delineate the mechanism driving AA-mediated cytotoxicity, whether this
effect is tissue-specific, and what represents a therapeutic dose of AA. Delivery of AA to the
tumour site, regulation of intracellular AA concentration, and limiting the potential off-target
effects are all challenges that remain to be overcome.

Screening assays will be required to determine the selectivity of new compounds in targeting
2OGDD family members, to avoid the significant potential risk of undesired side-effects by

Trends in Endocrinology & Metabolism, April 2018, Vol. 29, No. 4 255
inhibition of other 2OGDD-dependent processes [119,120]. However, concurrent inhibition or
activation of multiple 2OGDD pathways may be desirable in certain situations. As an example in
the treatment of cancer, combined activation of the PHD and TET enzymes may result in
reduced HIF-mediated angiogenesis and encourage cell differentiation, respectively, with the
overall effect of limiting malignant growth. Further work will be required to understand how to
specifically target malignant cells to maximise the benefits of these types of drug approaches.
Once optimised this approach may be relevant in the treatment of other common multifactorial
diseases with known mitochondrial involvement. Mitochondrial dysfunction may contribute to
pathology in these conditions by altering metabolic signalling through 2OGDD pathways and
thus may be amenable to treatments that redirect metabolic flux.

Finally, there is a need to explore the coordination of the 2OGDD SCP model in the context of
other metabolic–epigenetic signalling pathways, to predict more accurately the consequences
of metabolic programme switches (see Outstanding Questions). For example, the reductive flux
of 2OG to citrate and subsequent conversion to cytoplasmic acetyl-CoA may indirectly affect
histone acetylation. For further insight on the compartmental regulation of acetylation, the
reader is directed towards an excellent recent review [121]. An integrative study of metabolism
in cell-fate decision promises to have far-reaching potential, both in our understanding of cell
development and differentiation and the treatment of these processes when they become
dysfunctional.

Acknowledgements
P.F.C. is a Wellcome Trust Senior Fellow in Clinical Science (101876/Z/13/Z) and a UK National Institute for Health
Research (NIHR) Senior Investigator who receives support from the Medical Research Council Mitochondrial Biology Unit
(MC_UP_1501/2), the Medical Research Council (UK) Centre for Translational Muscle Disease (G0601943), and the NIHR
Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust and the University of
Cambridge. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the
Department of Health.

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