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Inhibition of pyruvate dehydrogenase kinase improves pulmonary arterial

hypertension in genetically susceptible patients

Authors: Evangelos D. Michelakis1*, Vikram Gurtu1, Linda Webster1, Gareth Barnes2,

Geoffrey Watson2, Luke Howard3, John Cupitt2, Ian Paterson1, Richard B. Thompson4,

Kelvin Chow4, Declan P. O’Regan5, Lan Zhao2, John Wharton2, David G. Kiely6, Adam

Kinnaird1, Aristeidis E. Boukouris1, Chris White7, Jayan Nagendran7, Darren H. Freed7,

Stephen J Wort8, J. Simon R. Gibbs3, Martin R. Wilkins2*.

Affiliations:1 Department of Medicine, University of Alberta, Edmonton T6G2B7, Canada.2 Department of Medicine, Imperial College London, London W12 0NN, UK.3 National Pulmonary Hypertension Service, Imperial College Healthcare Trust NHS, Hammersmith

Hospital, Du Cane Road, London W12 0NN, UK.4 Department of Biomedical Engineering, University of Alberta, Edmonton T6G2B7, Canada.5 Medical Research Council, London Institute of Medical Sciences, Imperial College London,

Hammersmith Hospital Campus, London W12 0NN, UK.6 Sheffield Pulmonary Vascular Disease Unit, Royal Hallamshire Hospital, Sheffield, S10 2JF, UK.7 Department of Surgery, University of Alberta, Edmonton T6G2B7, Canada.8 National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY

* Correspondence: EDM (em2@ualberta.ca) and MRW (m.wilkins@imperial.ac.uk)

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One Sentence Summary: Metabolic modulation with dichloroacetate improves hemodynamics in

genetically susceptible patients with idiopathic pulmonary arterial hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a progressive vascular disease with a high mortality rate. It is

characterized by an occlusive vascular remodeling due to a pro-proliferative and anti-apoptotic

environment in the wall of resistance pulmonary arteries (PAs). Proliferating cells exhibit a cancer-like

metabolic switch where mitochondrial glucose oxidation is suppressed while glycolysis is upregulated as

the major source of ATP production. This multifactorial mitochondrial suppression leads to inhibition of

apoptosis and downstream signaling promoting proliferation. We report an increase in pyruvate

dehydrogenase kinase (PDK), an inhibitor of the mitochondrial enzyme pyruvate dehydrogenase (PDH,

the gate-keeping enzyme of glucose oxidation) in the PAs of human PAH compared to healthy lungs.

Treatment of explanted human PAH lungs with the PDK inhibitor dichloroacetate (DCA) ex vivo

activated PDH and increased mitochondrial respiration. In a 4-month open-label study, DCA (3 to

6.25mg/kg bid) administered to patients with idiopathic PAH (iPAH) already on approved iPAH

therapies led to reduction in mean pulmonary artery pressure and pulmonary vascular resistance and

improvement in functional capacity, but with a range of individual responses. Lack of ex vivo and

clinical response was associated with the presence of functional variants of SIRT3 and UCP2 that predict

reduced protein function. Impaired function of these proteins causes PDK-independent mitochondrial

suppression and pulmonary hypertension in mice. This first-in-human trial of a mitochondria-targeting

drug in iPAH demonstrates that PDK is a druggable target and offers hemodynamic improvement in

genetically susceptible patients, paving the way for novel precision medicine approaches in this disease.

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Introduction

Pulmonary arterial hypertension (PAH) is a complex vascular disease leading to right ventricular

(RV) failure and death from a proliferative vascular remodeling that obstructs the lumen of resistance

pulmonary arteries (PA) (1). Approved therapies (phosphodiesterase-5 inhibitors, endothelin receptor

antagonists and prostanoids) act mainly as vasodilators and improve symptoms but fail to arrest or

reverse the disease (2). A cancer-like metabolic remodeling drives, at least in part, the structural changes

in the pulmonary vascular wall. Specifically, a suppression of mitochondrial glucose oxidation in all

cellular elements of the PAs, namely pulmonary artery smooth muscle cells (PASMC), endothelial cells

and fibroblasts, leads to inhibition of mitochondria-dependent apoptosis and secondary upregulation of

glycolysis promoting pro-proliferative signaling and direction of carbon sources toward cellular building

blocks rather than oxidation (3-9). Central to this is the inhibition of pyruvate dehydrogenase (PDH), the

gate-keeping enzyme of glucose oxidation, which catalyzes the mitochondrial production of acetyl-CoA

from pyruvate, the end-product of glycolysis, which in turn feeds the Krebs cycle to complete glucose

oxidation.

Several potential mechanisms of PDH inhibition have been described. Induction of pyruvate

dehydrogenase kinase (PDK) leads to its complexing with, and tonic inhibition of, mitochondrial PDH

by phosphorylation at serine-293. Whereas in healthy tissues PDK expression is low, the enzyme can be

induced selectively in the pulmonary circulation by the activation, even under normoxia, of hypoxia-

inducible factor 1 (HIF1), which is upregulated in PAH (10). PDH can also be inhibited by tyrosine

kinase phosphorylation at tyrosine-301 (11). Another mechanism is through inhibition of Sirtuin 3

(SIRT3), the main mitochondrial deacetylase, which activates several mitochondrial enzymes including

PDH; Sirt3 knockout mice develop spontaneous pulmonary hypertension and SIRT3 activity is decreased

in human PAH lungs (12). Last, inhibition of Uncoupling Protein 2 (UCP2), a mitochondrial protein that

regulates calcium entry, leads to a decrease in mitochondrial calcium (13, 14). Many mitochondrial

enzymes including PDH are calcium-dependent and Ucp2 knockout mice develop spontaneous

pulmonary hypertension (13, 15). Thus deficiency in SIRT3 and/or UCP2 inhibits PDH in a PDK-

independent manner.

Two relatively common single nucleotide polymorphisms (SNPs), rs11246020 for SIRT3 and

rs659366 for UCP2, have been reported to decrease the activity and expression of these proteins

respectively (16-18). Both gene variants are relatively frequent in the general population and have been

associated with metabolic syndrome in humans (16, 17, 19), which is highly prevalent in PAH patients

(20).

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PDK is a druggable target that has been effectively inhibited by the small molecule

dichloroacetate (DCA) in animal models and early-phase trials in cancer (21-25). DCA has also been

clinically tested, with a good safety profile, in patients with congenital mitochondrial diseases, including

PDH deficiency (26-29). We thus explored the role of PDK and DCA in human PAH with a series of

experiments using archived human lungs, PAH lungs studied ex vivo immediately after removal at

transplant surgery (ex vivo lung perfusion, EVLP) and an explorative open-label phase-I clinical trial in

iPAH patients. We also explored whether the presence of loss-of-function gene variants for SIRT3 and

UCP2 provide resistance to the ability of DCA to activate PDH and reverse mitochondrial suppression in

human PAH.

Results

PDK expression is increased in the pulmonary arteries of human PAH lungs

We measured the expression of the two ubiquitously expressed PDK isoforms (1 and 2) (30) and

PDH (Fig. 1A) in lung tissues from 10 patients (6 with PAH and 4 non-PAH controls, (Table 1) by

confocal microscopy and immunoblotting. PDK protein was markedly increased in the wall of small

muscularized pulmonary arteries (PA) from PAH compared to non-PAH lungs (Fig. 1B and fig. S1).

This upregulation was strong in PA smooth muscle cells (PASMC) co-stained with smooth muscle actin

antibody but was also apparent throughout the vessel wall in other PA elements, for example in

endothelial cells, fibroblasts and tissue inflammatory cells. This is consistent with animal studies that

show that these cell types in PAH undergo a metabolic remodeling (suppressed glucose oxidation and

upregulated glycolysis) (7, 8) similar to that of PASMC (4, 5). Immunoblots from tissue samples taken

from the periphery of lungs, which are enriched with small PAs, confirmed the upregulation of both PDK

isoforms in PAH (Fig. 1C). This was associated with phosphorylation of serine-293 on the PDH-E1

subunit, marking PDH inhibition. We concluded that PDK upregulation is an active component of

metabolic remodeling in human PAH and that the selectivity of this upregulation to diseased tissues

makes PDK a potential therapeutic target for PAH.

DCA increases PDH activity and mitochondrial function in human PAH lungs ex vivo

We then studied mitochondrial function in 5 human lungs (Table 1) explanted at transplant

surgery using EVLP under conditions (pH, temperature, perfusion, ventilation) that best mimic the in-

vivo environment. The lungs were perfused and ventilated upon removal (Fig. 2A) and biopsies were

taken from peripheral sites of each lung, which are enriched with resistance PAs, as soon as baseline

conditions were established at 37C. After one hour of perfusion with DCA at a concentration similar to

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the trough concentrations reported in patients on oral DCA therapy, additional biopsies were taken and

pre-post comparisons were made. PDH activity was measured with two standard techniques: a

biochemical dipstick assay based on the PDH-driven production of NADH (Fig. 2B) and immunoblots

measuring serine-293 PDH-E1 phosphorylation (Fig. 2C). We also measured mitochondrial respiration

in these tissues using a Seahorse protocol (Fig. 2D). Variability in the response to DCA was observed.

Lungs 1, 2 and 3 showed an increase in PDH activity and mitochondrial respiration, whereas lung 4 was

resistant to DCA and showed a small decrease in respiration during DCA perfusion. Lung 5 (control) was

perfused with vehicle and, like lung 4, exhibited no change in PDH activity and a small decrease in

respiration during the same perfusion period. We speculated that non-PDK dependent mechanisms, like

the presence of reduced SIRT3 and/or UCP2 activity, may impair the response to DCA.

Improvement in hemodynamics and functional capacity in iPAH patients after chronic oral DCA

administration

To investigate the therapeutic potential of PDK inhibition with DCA in vivo, a proof-of-concept

4-month, dose-finding trial of 3, 6.25 or 12.5 mg/kg by mouth twice daily (bis in die, b.i.d.) was

conducted in patients clinically stable on treatment with approved iPAH therapies (clinicaltrials.gov

NCT01083524). Of the 23 patients screened, 20 satisfied the inclusion/exclusion criteria and were

enrolled in the study (Table 2). All patients were on sildenafil, 13 on dual therapy (sildenafil +

endothelin receptor antagonist) and one on triple therapy (sildenafil, endothelin receptor antagonist and

parenteral prostanoid) in addition to background therapies. No patient had initiated PAH-approved

therapies for a minimum period of 6 months prior to enrollment, and maintenance dose was stable during

this time except for patient #12 (Table 2), who had an increase in the dose of an endothelial receptor

antagonist 4.4 months prior to enrollment. This patient, like all others, was stable with no signs of

clinical improvement or worsening of disease for at least 8 weeks prior to enrollment (an eligibility

requirement).

There was no clinically significant change in the QT interval of the electrocardiogram, cardiac

rhythm, liver, bone marrow or renal function. Dose-limiting toxicity, specifically paresthesia affecting

the dorsum of the foot, toes and fingers (grade II peripheral neuropathy), consistent with a previously

described reversible and dose-dependent, non-demyelinating peripheral neuropathy (26, 28), developed

in all 5 patients taking the highest dose tested (12.5 mg/kg b.i.d.). Four patients withdrew from the study

at that point (weeks 3 to 11) and one accepted a protocol-driven decrease in the dose to 6.25 mg/kg b.i.d.,

with improvement of symptoms within 1-3 months in all patients. Thus all patients completing the

protocol (n=16) were taking 3 or 6.25 mg/kg b.i.d., and 6.25 mg/kg b.i.d. was established as the highest

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tolerated dose, consistent with previous studies in patients with genetic mitochondrial diseases and

cancer (23-26). No patient deteriorated clinically or exhibited a decrease in the 6-minute walk of more

than 10% from baseline and no patient required hospitalization during the study. There were no changes

in PAH-approved and background therapies during the study.

Exposure to DCA led to an improvement in hemodynamic measurements at right heart

catheterization [mean PA pressure (mPAP, P < 0.05), pulmonary vascular resistance (PVR, P < 0.05)]

and functional capacity (6-minute walk test, P < 0.05) in the cohort as a whole, but inter-individual

variability in response was apparent (Fig. 3A). Seven patients responded to DCA with a decrease in

mPAP greater than 5 mmHg (mean decrease 9.1 mmHg) with an increase or no change in cardiac output

(highlighted box in Fig. 3B); mean PVR decreased by 11832 dyn.s.cm-5 and 6-minute walk increased

by 5318m in this group. Considering that these patients were already treated with PAH therapies, this

response is clinically meaningful and of a magnitude comparable to approved PAH therapies, which have

shown a mean decrease in mPAP of less than 5 mmHg in landmark trials of previously untreated patients

[epoprostenol -4.5 mmHg (31), bosentan -1.6 mmHg (32), sildenafil -2.1 mmHg (33)].

To better understand the variation in response, we first looked at plasma DCA concentrations

(Fig. 4). Mean trough concentration in both the 3mg/kg bid (0.17 mM) and 6.25 mg/kg bid (0.59 mM)

treatment groups demonstrated adequate exposure of PDK to DCA [the Ki of DCA for PDK2 is 0.2 mM

(30)] and there was no dose-response relationship with respect to change in mPAP or PVR. The mean

trough DCA concentration in the seven patients with a good response to DCA was 0.22 mM compared to

0.54 mM in the “non-responder” group, excluding underexposure to DCA as an explanation of the lack

of response.

Differences in the response to DCA are associated with SIRT3 and UCP2 variants

We speculated that the lack of response to DCA in both the clinical trial and the EVLP

experiments has a genetically-driven biochemical basis and is associated with the presence of functional

variants in SIRT3 and UCP2. As discussed earlier, the presence of these variants may cause a PDK-

independent inhibition of PDH, which would not be responsive to DCA.

The SIRT3 rs11246020 “A allele” is associated with a point mutation that causes a change of

valine to isoleucine at residue 208 within the conserved catalytic deacetylase domain, causing a 34%

decrease in SIRT3 activity compared to the “G allele” (17). The UCP2 rs659366 “G allele” affects the

promoter region of the gene, causing a decrease in transcription and decreased mRNA levels compared

to the “A allele” (18, 19). Although SIRT3 and UCP2 are both in chromosome 11, they are not in linkage

disequilibrium. There is evidence for a “gene dose-response” with both variants: Sirt3-/- mice show

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greater inhibition of enzymatic activity and worse pulmonary hypertension compared to Sirt3+/- mice

(12); and UCP2 mRNA expression is associated with the presence of the G allele, with the GG, GA and

AA alleles associated with progressively increased mRNA expression (18). We thus developed an SNP

score where 1 point is given for each loss-of-function variant present: an SNP score of 0 means that both

the variants are absent, a score of 4 denotes both variants are present in a homozygous manner and scores

in between represent the sum of points for each allelic risk variant present (table S1). A high SNP score

predicts inhibition of PDH independent of PDK and greater resistance to DCA. Indeed, lung 4, which

exhibited resistance to DCA, had an SNP score of 3, whereas lungs 1-3, which responded to DCA, had

SNP scores of 0 or 1 (Figs. 2B-D, Table 1).

Consistent with the ex vivo experiments, there was an inverse relationship between the SNP score

and change in mPAP, PVR and 6-minute walk test: the lower the SNP score, the stronger the response to

DCA (Table 2 and Fig. 3B). While the presence of each gene variant alone was inversely but weakly

associated with a decrease in mPAP (fig. S2), their combined presence (fig. S3) exhibited a stronger

relationship with the change in mPAP, suggesting an additive genetically-driven resistance to DCA. A

similar pattern characterized the response of RV function, the major determinant of morbidity and

mortality in PAH. RV ejection fraction, measured by cardiac MRI, improved in patients with an SNP

score of 0 (p<0.05 compared to their baseline), whereas the patients with SNP scores of 1 had variable

responses and those with higher SNP scores showed no improvement (fig. S4).

Exploratory imaging biomarkers support the clinical response to DCA

We used two imaging biomarkers in patient subsets to assist with biological interpretation of the

response to DCA. Using gadolinium lung perfusion imaging with MRI we found that the responders with

a decrease of mPAP >5mmHg had a ~150% increase in lung perfusion compared to the non-responder

group (mean decrease in transit time -0.55 versus -0.22 seconds, respectively, fig. S5A). We also

measured parenchymal lung glucose uptake by 18fluoro-deoxyglucose positron emission tomography and

computed tomography (18FDG-PET-CT). Consistent with increased glycolysis, animal models and

patients with PAH exhibit higher lung glucose uptake as measured by lung 18FDG-PET compared to no-

PAH controls (8, 34). By increasing glucose oxidation, DCA is expected to reduce glycolysis and thus

glucose uptake. The hemodynamic responders showed an overall decrease in 18FDG uptake, whereas the

non-responders showed an increase (fig. S5B). Consistent with the relationship between hemodynamic

response to DCA and SNP score, a low SNP score was associated with a reduction in 18FDG uptake in

response to DCA, whereas a high SNP score was associated with an increase in 18FDG uptake. Examples

of two responders are shown in Fig. 5.

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Discussion

In the first attempt to target a mitochondrial enzyme to treat humans with vascular disease, we

report that DCA in doses up to 6.25 mg/kg po bid over 4 months are generally well-tolerated and can

result in a significant reduction in mPAP and PVR in iPAH. PDK inhibition by DCA increases PDH

activity and oxygen consumption in isolated perfused/ventilated PAH lungs, consistent with

improvement in mitochondrial function.

DCA has a very specific mechanism of action: mimicking pyruvate’s structure, it competes with

pyruvate for its binding “pocket” in PDK and thus mimics the “end-product inhibition” that results from

pyruvate-PDK binding (35). This has been confirmed by crystallization studies of PDK with pyruvate or

DCA in situ (36). In addition, molecular (small interfering RNA-induced) inhibition of PDK2 in cancer

cells mimics DCA, which does not exert any additional effects in cells lacking PDK (21). PDK2 is

induced in the lungs of PAH patients and of all the PDK isoforms, PDK2 is the most sensitive to DCA

(30).

Poor DCA responders were characterized by a biochemical resistance which was associated, both

in the ex vivo experiments and the clinical trial, with the presence of variants in SIRT3 and UCP2 that

predict reduced protein activity. We propose that PDH inhibition in these patients is less dependent upon

PDK. Specifically, DCA may inhibit PDK but fail to increase PDH activity and respiration in patients in

whom PDH is also inhibited by increased acetylation and/or lack of mitochondrial calcium in vascular

cells due to SIRT3 and UCP2 gene variants that inhibit SIRT3 activity and decrease UCP2 expression.

The correlation between the SNP score and clinical response to DCA was a post-hoc analysis.

But unlike randomized trials where a post hoc exploration of a biomarker can introduce bias, this concern

does not apply in studies of genetic variants with known effects that are not in linkage disequilibrium.

The random distribution of the relevant variants during miosis reduces bias and “Mendelian

Randomization” is being increasingly used to dissect genotype-response relationships (37). Post hoc

analysis of genetic factors causing resistance to a drug has been used successfully in cancer, leading to

conclusions strong enough to change clinical practice, without evidence from prospective studies (38,

39). Our hypothesis-driven selection of these gene variants was based on strong preclinical studies,

which directly link the function of these genes with PAH pathogenesis. Our sample size is too small to

evaluate whether these variants are enriched in PAH compared to healthy populations. Nevertheless, a

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gene variant may not be enriched in a population with the disease but may still affect the response to

therapy.

We used non-invasive imaging biomarkers, 18FDG-PET-CT and MRI lung perfusion, to aid the

biological interpretation of the hemodynamic responses. These tools may not be necessary to assess

response in future studies but it would be of interest to know whether patients exhibiting high lung 18FDG uptake are more responsive to DCA in future trials. An increase in lung perfusion shown in the

gadolinium-MRI studies, even in areas not perfused prior to DCA therapy (Fig. 5A), is consistent with

potential regression of occlusive vascular remodeling.

Previous studies in animals and humans have described an inotropic effect of DCA on the RV,

perhaps due to a shift in the utilization of metabolic substrate (carbohydrates vs fatty acids) (40, 41). The

combined effect of a decrease in RV afterload due to regression of pulmonary vascular remodeling and

RV inotropy is clinically very attractive. The long-term direct effects of DCA on RV remodeling are not

known but do not appear to be adverse, considering that none of our patients deteriorated clinically. We

did detect an increase in RV ejection fraction in DCA responders, but did not detect a decrease in RV

hypertrophy (fig S4). This is not necessarily a disadvantage. Given that the RV afterload is still high in

DCA responders, the maintenance of a compensating RV state (hypertrophy) is beneficial, compared to a

potential transition to a decompensated state of RV dilatation and failure (42).

Activated immune cells play a paramount role in the pathogenesis of PAH (43) and activation of

these cells is associated with a suppression of glucose oxidation and switch to glycolysis (44). Although

the role of PDK in these cells is not known, it is possible that DCA may have effects on these cells,

limiting their activation and the inflammatory burden in PAH – a possibility that requires further studies.

PDK is induced by HIF and this has been confirmed in both cancer and PAH models, where HIF

activity is increased even under normoxia (10, 45). A primary mitochondrial suppression can lead to

secondary activation of HIF, completing a positive feedback loop (12, 25, 46) (Fig. 1A). There are many

potential mechanisms that can contribute to primary mitochondrial suppression in PAH (including the

presence of SIRT3 and UCP2 variants), leading to a positive feedback loop between mitochondrial

suppression and HIF activation, where PDK plays a central role (5). PDK inhibition can break this

feedback loop and reverse the metabolic remodeling, promoting apoptosis and inhibiting proliferation

within the PA wall.

Other molecular factors contributing to the metabolic reprogramming in PAH could contribute to

a biochemical resistance to mitochondrial activators. In the case of DCA, these include a decrease in

SIRT3 protein abundance by non-genetic mechanisms (12) or a potential decrease in the mitochondrial

calcium uniporter (MCU), an important calcium entry regulator in mitochondria, recently found to be

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important in PAH (47). UCP2 has been shown to regulate the function of the MCU (14). However the

identification of these potential molecular causes of DCA resistance in patients requires lung biopsies,

which are contraindicated in PAH patients. On the other hand, the gene variants that we studied here can

be easily detected in many tissues, including blood, making them attractive tools for future precision

medicine trial designs. Lastly, there is some evidence that SNPs in glutathione transferase zeta

1/maleylacetoacetate isomerase lead to hyper- or hypo-metabolizers of DCA, at least in children (48).

Although there does not appear to be a relation between the clinical response and DCA trough

concentration in our small cohort (Fig. 4), we did not measure DCA metabolites and the possibility that

such SNPs may be relevant should be studied in larger cohorts of future studies.

This work translates to humans the knowledge accumulated from multiple preclinical studies and

supports the newly proposed metabolic theory of PAH (4, 5). The beneficial hemodynamic effects of

DCA were observed in patients already taking licensed medications for PAH. These agents are not

known to have any effect on PDH activity and our observations suggest that the decreased PDH activity

is an unexploited therapeutic target in clinical practice. A factor to consider in future clinical trials of

DCA is combination with a tyrosine kinase inhibitor since tyrosine kinases can both inhibit PDH by

phosphorylating a site different than that of PDK(11), as well as activate PDK by phosphorylating PDK

itself (49). We suggest that genotype should be considered as a basis for stratification in future clinical

studies of DCA or other metabolic modulators, with a precision medicine design, either ensuring

randomization of patients according to their SIRT3/UCP2 SNP score or excluding patients with a SNP

score of >1 from the study, to maximize detection of a beneficial response.

Materials and Methods

Study design: This work consists of a preclinical component, where the effects of DCA were tested on

human PAH lungs ex vivo and the amount of its target enzyme (PDK) was measured in human PAH and

control lungs, and a clinical component where a phase-1, dose discovery trial (NCT01083524) was

conducted to measure the effects of DCA on several clinical parameters in patients with iPAH. The

investigators performing the experiments were blinded for the preclinical data but the clinical study was

open label.

Human tissues samples: The protocols for clinical and human tissue studies were approved by the

human studies ethics committees at the University of Alberta (Edmonton, UK) and Imperial

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College/Hammersmith Hospital (London, UK). Human tissues were processed and studied under

identical tissue handling and staining protocols (Table 1).

Mitochondrial respiration measurements: Lung biopsies were taken before and after treatment with

DCA during EVLP for further analysis of oxygen consumption using the Seahorse XFe 24 Analyzer

(Agilent Technologies). Tissue was cut into segments weighing approximately 5 mg and immediately

plated onto an XF24 Islet Capture Microplate, with islet capture screens to secure the tissue at the bottom

of the well, and submerged in the EVLP perfusate. Oxygen consumption rate was measured and

normalized to mass of tissue per well.

PDH activity analysis: PDH activity was measured with a commercially available MitoProfile Dipstick

assay kit (Mitosciences) as previously described (50, 51). 200 µg of protein were incubated with the

dipstick containing the PDH complex antibody. With this method, the PDH complex is immunocaptured

and the production of PDH-driven NADH is measured.

Immunoblots: imunoblots were performed with SDS-PAGE as previously described and analyzed using

ImageJ (NIH) (12, 25). Antibodies: PDK1 (Abcam, ab110025; detected at 49 KDa), PDK2 (Abcam,

ab68164; detected at 46 KDa), PDH-E1 (Abcam, ab168379; detected at 43 KDa), phospho-Ser293-

PDH-E1 (EMD Millipore, AP1062; detected at 44 KDa), and actin (Abcam, ab3280; detected at 42

KDa). The PDK-PDH antibodies have been validated in a recent publication (52).

Confocal Imaging: Immunofluorescence staining was performed as previously described and imaging

was done using a Zeiss LSM-510 NLO model (Carl Zeiss) (12, 25, 52). Antibodies used were PDK1

(Abcam, ab110025), PDK2 (Santa Cruz, sc-14484) and alpha smooth muscle actin (Abcam, ab5694).

SNP genotyping assay: Genomic DNA was extracted from buffy coat using Allprep RNA/DNA kits

(QIAGEN). DNA samples were quantified with a Nanodrop Spectrophotometer (ND-8000) and

normalized to a concentration of 50 ng/μL (diluted in 10mM Tris/1mM EDTA). Samples were

genotyped by TaqMan® SNP Genotyping Assays for rs11246020 and rs659366 (Applied Biosystems)

and processed in triplicates according to standard protocol and read on the ABI Prism 7900 Sequence

Detector (Applied Biosystems) (12).

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Ex Vivo Lung Perfusion (EVLP): EVLP allowed the perfusion and ventilation of human lungs from

donors and recipients at transplant surgery. The perfusate entered the pulmonary artery via a tube,

controlled by a pump in combination with pressure and flow-sensing catheters. The bronchi were

connected with tubes to an ICU ventilator. The efflux from the pulmonary veins entered a de-oxygenator,

returning de-oxygenated blood into the pulmonary arteries, mimicking in vivo conditions. Flow wass

initiated slowly in a retrograde fashion to remove air from the circuit. Anterograde flow started at 150

ml/min with an albumin-based solution supplemented with Na+ 138 mmol, K+ 6 mmol, Mg 2+ 0.8 mmol,

Cl- 142 mmol, SO4-2 0.8 mmol, H2PO4 0.8 mmol and glucose 5 mmol. The perfusate temperature was

increased to 37°C and flow rate was gradually increased to 40-50% of the estimated patient cardiac

output. A protective mode of ventilation was applied with a tidal volume of 7 ml/kg at 7 breaths per

minute, PEEP of 5 cmH2O, and FiO2 of 21%. The lungs were periodically expanded with inspiratory

holds to an airway pressure of 20 cmH2O. pH, pCO2, electrolytes and glucose were maintained at

physiologic concentrations with frequent measurements. After baseline conditions were established,

0.7mg/ml DCA (sodium salt, TCI America) was added to the perfusate. Peripheral lung biopsies were

obtained before and after DCA perfusion. The recipient’s lungs were obtained at the time of lung

transplantation and transferred immediately (within 5 minutes) to the laboratory for EVLP, under a

patient consent and protocol approved by the Institutional Review Board at the University of Alberta.

See Fig. 2A for EVLP details.

Clinical Protocol (NCT01083524): Adult patients with idiopathic PAH (iPAH) diagnosed by

recognized criteria (53) were treated with DCA for 4 months (Table 2). All were receiving background

and approved PAH therapies and had to be clinically stable with no dosing changes for at least 8 weeks

prior to enrollment (an eligibility requirement). Although initially enrollment of patients in WHO

functional Class III-IV was planned, the protocol was subsequently amended to also allow enrollment of

patients in Class II. Exclusion criteria included concomitant diabetes, chronic kidney and liver disease

and a history of neuropathy. There were 3 screen failures out of 23 patients screened. All patients signed

informed consents approved by the local human ethics committees. Three doses of DCA were tested: 3

mg/kg po b.i.d., 6.25 mg/kg po b.i.d.and 12.5 mg/kg po b.i.d.. Starting from the lowest dose, at least 3

patients in each site (Canada and United Kingdom) had to be treated without evidence of toxicity for 4

weeks before another patient could be enrolled to the higher dose. Since the main anticipated toxicity,

peripheral neuropathy, was previously described to be a dose-dependent and reversible, non-

demyelinating neuropathy, a decrease in the dose was allowed for the 2 higher doses if symptoms

occurred. Pathogen-free DCA (sodium salt) was purchased from TCI America. Pharmacists placed the

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individual doses of DCA powder (according to patient weight) in individual 20 cc sterile light-proofed

vials and patients added water to each bottle and dissolved DCA, which is highly water soluble, before

drinking it.

The primary end-point was safety and tolerability and secondary end points included change from

baseline in a) mPAP and PVR measured by standard right heart catheterization, b) functional capacity

measured by the 6-minute walk test, and c) RV mass and ejection fraction measured with standard

cardiac MRI protocols. We also assessed two exploratory endpoints: d) the biochemical response to DCA

using lung 18FDG-PET-CT (London site) as a measure of glucose uptake in the lungs, since it was

hypothesized that a DCA-induced activation of glucose oxidation would result in suppression of

glycolysis and thus a decrease in glucose uptake; and e) MRI-measured lung perfusion, measuring the

transit time of gadolinium through the pulmonary circulation, as it was hypothesized that a DCA-induced

regression of pulmonary vascular remodeling would increase lung perfusion (decreasing gadolinium

transit time) even in areas not perfused prior to DCA therapy (more than increasing the perfusion in

already perfused areas). The safety visits, which included blood tests for renal, hepatic and bone marrow

toxicity, ECG, physical examination and 6 minute walk distance, were performed monthly. Right heart

catheterization, MRI and PET studies were performed at baseline and at 4 months. There were no

patients lost to follow up. The protocol was terminated when the dose limiting toxicity was clearly

reached.

MRI studies: RV mass and volumes were measured using a standard approach by two readers blinded to

the patient’s background or state (pre versus post DCA). Lung perfusion was studied with three-

dimensional dynamic contrast-enhanced MRI using 1.5T Siemens Sonata systems. Typical scan

parameters included 0.54 ms echo time, 1.5 ms repetition time, 15° flip angle, 192x72x20 matrix with a

400x300x150 mm field of view (axial orientation), rate 2 parallel imaging (GRAPPA), with a temporal

resolution of 1.24 seconds per image, and a total of 30 acquired images. All subjects were injected with

0.1mmol/kg of gadopentetate dimeglumine (Magnevist, Bayer) at a rate of 5 ml/s followed by a 20 ml

saline flush. Injections were timed to ensure 5 baseline (non-contrast) image acquisitions. Subjects were

instructed to hold their breath at end-expiration.  MRI studies were not possible on patient 16 for the RV

mass and ejection fraction protocols (patient declined) and on patients 8, 11 and 15 for the perfusion

studies due to motion artifacts (Table 2). The underlying theory and application of contrast-enhanced

dynamic perfusion in the lungs has previously been described (54, 55). Briefly, signal intensity curves

from the lung tissue and blood pool (main trunk of the pulmonary artery, arterial input function) were

first converted to contrast agent concentration using a Bloch-equation look-up table approach. The tissue

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time-intensity curves were subsequently used to estimate the mean transit time (MTT) of the contrast

agent in the lung parenchyma, using an exponential de-convolution approach to account for the effects of

the arterial input function. MTT values were calculated in each pixel, and the average values over the

entire lung are reported, with exclusion of pixels with contributions from larger arteries or veins.   

PET-CT studies: Lung 18FDG (120 MBq) distribution was measured 1.5 hours following intravenous

administration as previously described (34). Regional glucose metabolic rate was calculated from the

PET image data and from blood samples taken during the scan. The lung region of interest was identified

from the CT image and the rate of change of activity was calculated for each voxel. Blood rate was

plotted against image rate for each lung voxel to produce a plot, which tends to a straight line (Patlak

plot), the slope of which gives rate of tissue uptake. The Patlak slope was calculated for every voxel in

the lung region. A voxel score was then calculated by dividing by the CT-derived tissue density and

scaling by 1000, giving an index of the glucose metabolic rate. Histograms of the voxel score distribution

were plotted as “before-after” pairs and a leftward shift in distribution indicates a reduction in 18FDG

uptake (Fig. 5). The mode of the voxel score histogram was found by smoothing data with a Gaussian

filter and searching for the peak. The PET-CT studies were only performed at the London site (n=7).

Trough DCA concentrations: Plasma samples were prepared using a DSS solution (0.5 mM) as an

internal chemical shift reference. The samples themselves consisted of 250µl of 90% plasma and 10%

standard internal reference and were placed in Norell S-3-200 3mm NMR tubes. NMR data were

collected on an Agilent 700 MHz (16.45T) NMR spectrometer equipped with a VNMRS 4-channel

console as previously described(52). The system had a cryogenically cooled inverse triple resonance

(HCN) probe containing a Z-pulsed field gradient coil. An Agilent 7620 sample-handling robot was

utilized. Samples were run at 25°C and individually tuned and matched via an Agilent ProTune module.

Each spectral was automatically phased and manually corrected to reduce baseline distortion due to

residual solvent signal. The spectra were referenced to DSS, and then re-phased and re-referenced for

each sample to confirm consistency. Regions for integration were 8.6364-6.5572, 6.1000-6.000, 5.9682-

5.6456, 5.4766-5.1386, 5.0259-0.5397 and 0.1200-0.1200 PPM. Regions not included in the integration

were utilized to apply a standard VNMRJ baseline correction. The 6.1 to 6 and 0.12 to -0.12 integrated

regions were subsequently used for DCA to DSS quantitation calculation, respectively.

Statistical analysis was performed using IBM SPSS, Version 24.0 (Armonk, NY: IBM Corp). Data are

shown as means with error bars indicating standard error of the mean. All data undergoing statistical

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analysis were subjected to Shapiro-Wilk test of normality. Some of the groups in Fig. 1 did not follow a

normal distribution. Thus, mean data from immunoblots (Fig. 1) were compared with the non-parametric

Mann-Whitney U test.  Mean differences (pre-post DCA) from clinical parameters (Fig. 3, fig. S4) did

not deviate from normality, therefore mean differences were compared via the parametric paired t-test.

For correlation of SNP score to clinical parameters, the Spearman rank correlation was chosen given the

small sample size in some SNP categories. Spearman rank correlation coefficient is shown (rS) with

respective p-values in Fig. 3 and fig. S4. Significance for all statistical testing was determined to be

P < 0.05.

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Acknowledgments: We are grateful to the physicians and staff of the University of Alberta Pulmonary Hypertension Program and the Hammersmith and Sheffield Pulmonary Hypertension Service for assistance with the recruitment and investigation of patients. We are indebted to the patients for their participation in the study.Funding: This paper presents independent research that was funded by the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada and the Hecht Foundation (Vancouver, Canada) to EDM; and a joint Medical Research Council and British Heart Foundation grant (G0701637) and a Wellcome Trust – GSK programme to MRW. This work was supported by the Mazankowski Alberta Heart Institute (Edmonton, Canada) and the National Institute for Health Research (NIHR)/Wellcome

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Trust Imperial Clinical Research Facility at Imperial College Healthcare NHS Trust (London, UK). Author contributions: EDM conceived the study. EDM and MW designed, funded and supervised the study. EDM, VG and MW analyzed the data. EDM wrote the first draft, which was edited by MW and VG. All the authors contributed to the acquisition of either the preclinical or clinical data, co-edited and approved the first and subsequent drafts and revisions of the paper.Competing interests: noneData and materials availability: all of the data are included in the paper or SM itself.

Figure legends

Fig. 1. PDK is important in the metabolic remodeling of PAH and is upregulated in human PAH lungs.A. Schematic demonstrating how PDK and SIRT3 and UCP2 suppress mitochondrial function by independent mechanisms (inhibition of PDH and glucose oxidation, global increase in mitochondrial acetylation, global decrease in mitochondrial calcium, respectively). This mitochondrial suppression inhibits apoptosis and promotes proliferation, leading to the proliferative vascular remodeling characteristic of PAH. DCA is a selective PDK inhibitor and does not affect other mechanisms of PDH inhibition. DCA may inhibit the feedback loop that links PDK-driven mitochondrial suppression and secondary normoxic activation of HIF1α to HIF-induced PDK induction. ETC: electron transport chain; Ca++

mito : intra-mitochondrial calcium. B. Confocal immunohistochemistry of a PAH and a non-PAH control lung with parallel staining for PDK1, PDK2, smooth muscle actin (SMA) and DAPI (a nuclear marker). Yellow boxes on differential interference contrast (DIC) images focus on small resistance pulmonary arteries, shown at higher magnification (right). An example of the stain only with secondary antibody is shown at the bottom. See Table 1 for patient information. DAPI: 4',6-diamidino-2-phenylindole. C. Immunoblots of peripheral lung tissues from PAH and control (non-PAH) lungs (for patient info see Table 1) probed for p-PDH-E1α, PDH-E1α, PDK1, and PDK2. Quantification of the PDK1 or PDK2 over actin and p-PDH-E1α/ PDH-E1α (normalized to actin) in each gel is shown. * P < 0.05, Mann-Whitney U test.

Fig. 2. DCA activates PDH and increases respiration in human PAH ex vivo lung perfusion (EVLP). A. A photograph and schematic of the custom-made EVLP system with a human lung (see methods). B-C. PDH activity measured from lung biopsies before & after one hour of DCA perfusion (0.7mg/ml). B. PDH activity dipstick assay, measuring NADH production from the PDH reaction (see box on top), quantitation (AU, arbitrary units), and SNP scores. C. Immunoblots for p-PDH-E1α and quantitation ([p-PDH-E1α/actin]/ [total PDH-E1α/actin]). Lung 1, 2, 3 showed an increase in PDH activity by DCA whereas Lung 4 was resistant to DCA (for patient info see table S1). The control lung 5 (see Table 1) was perfused with vehicle (perfusate) for the same time as lungs 1-4. D. Mitochondrial respiration (oxygen consumption) measured using a Seahorse protocol from pre- and post-perfusion biopsies. Top: an example of oxygen consumption rate (slope of each line) of a pre- and

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post-DCA perfusion biopsy from Lung 3. Bottom: the % change in oxygen consumption rate after DCA for all lungs. Perfusion of lung 5 (see table S1) with vehicle (perfusate) was a time control experiment.

Fig. 3. The effects of DCA on hemodynamic and functional endpoints and their association with genetic factors (variants of the SIRT3 and UCP2 genes) that cause resistance to DCA. A. mPAP, PVR, and 6-min walk performance of DCA-treated patients. Statistical significance was assessed using before-after paired t-test (P < 0.05). The meanSEM before and after DCA treatment and the P values are shown in each graph. B. Scatter plots of the DCA-induced changes in mPAP, PVR and 6-min walk, separated by the SNP score status of each patient. Statistical significance was assessed using a Spearman’s correlation test and rs and P values are shown in each graph. Highlighted region identifies 7 patients that exhibited a significant decrease in the mPAP (>5 mmHg decrease) associated with an increase or no change in the cardiac output, suggestive of a clinically meaningful response. Plots of the DCA-induced change in mPAP, PVR, and 6 min walk (meanSEM) over the SNP score are shown on the right.

Fig. 4. Trough serum DCA concentrations. Top: DCA concentration (meanSEM) in patients exposed to 6.25 mg/kg bid compared to 3mg/kg bid. Bottom: DCA concentration (meanSEM) in “responders” (decrease in mPAP by more than 5 mmHg) compared to “non-responders” patients.

Fig. 5. Examples of functional imaging biomarkers in two DCA responders. A. MR images showing gadolinium transit time before and after DCA and resolution of the D-shaped septum (arrow) in the heart of Patient #3. Note the color scale of the gadolinium transit time: blue indicates short transit time and increased perfusion; yellow/red indicates long transit time and little/no perfusion at baseline. B. PA pressure recordings and 18FDG uptake pre- and post-DCA from right heart catheterization and 18FDG-PET-CT, respectively, of Patient #10. A shift to the left on the distribution of the 18FDG uptake suggests a decrease in glucose uptake due to decreased glycolysis. Color heat maps show Patlak slope per gram of tissue (score) per voxel in lung sections, indicating the density of high-value voxels (coloured in yellow and red, indicating areas of high glucose uptake), overlaid on corresponding CT images.

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Table 1. Characteristics of patients offering lung tissues

C:

control tissue, P: patient tissue, EVLP: ex vivo lung perfusion, aPAH: associated PAH (collagen vascular disease), iPAH: idiopathic PAH, PDE5i: phosphodiesterase type 5 inhibitor, ERA: Endothelin Receptor Antagonist, Prostn: parenteral Prostanoid, sGC: soluble Guanylate Cyclase

22

patient Diagnosis age sex WHO

class PAH therapy

His

tolo

gy -

Imm

unob

lots

C1 lobectomy (tumor) 56 M II none

C2 unused transplant donor 25 M I none

C3 unused transplant donor 38 F I none

C4 unused transplant donor 41 F I none

P1 aPAH 34 F III PDE5i + ERA

P2 iPAH 18 M IV PDE5i + Prostn

P3 iPAH 39 F IV ERA + Prostn

P4 iPAH 31 F III ERA + Prostn

P5 iPAH 42 F III PDE5i + ERA + Prostn

P6 aPAH 40 M IV PDE5i + ERA + ProstnSIRT3varian

t

UCP2variant

EVLP

Lun

g

1 aPAH 62 F IV PDE5i + ERA + Prostn G/G A/A

2 aPAH 61 F IV PDE5i + ERA + Prostn G/A A/A

3 iPAH 32 F IV PDE5i + ERA + Prostn G/G A/A

4 iPAH 43 F IV PDE5i + ERA + Prostn G/A G/G

5

Chronic thromboembolic

pulmonary hypertension

60 M IV sGC stim + Prostn G/A A/A

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Table 2

Table 2. Baseline characteristics of enrolled patients

Pt: patient, RAP: right atrial pressure, PAWP: pulmonary artery wedge pressure, CO: cardiac output, Tx: therapy, P: phosphodiesterase type 5 inhibitor, E: Endothelin Receptor Antagonist, Pros: parenteral prostanoid. All patients diagnosed with idiopathic pulmonary arterial hypertension. ¹Dose decreased from 12.5 mg/kg. Patients 17-20 were withdrawn from the protocol due to a grade II peripheral neuropathy. Patients 1-16 completed the protocol and are all included in the Fig. 3 data analysis.

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pt age sex RAPmmHg

mPAPmmHg

PAWPmmHg

COL/min

6 minwalk

WHO class

PAH Tx

SIRT3 variant

UCP2 variant

SNPscore

DCA(mg/kg

bid)1 67 F 5 43 6 8.9 298 III P G/G G/A 1 3

2 54 F 3 50 5 3.4 450 II P+E G/G G/A 1 3

3 43 F 20 55 9 5.6 366 III P+E G/G A/A 0 3

4 60 F 4 62 11 3.5 409 II P+E G/G A/A 0 6.25

5 27 F 8 56 7 4.3 533 II P+E A/A A/A 2 6.25

6 63 F 6 33 10 6.1 385 III P+E G/G G/G 2 6.25

7 54 F 7 46 11 4.7 407 II P+E G/A A/A 1 6.25

8 75 M 13 48 13 6.7 262 III P G/G A/A 0 6.25

9 73 M 8 45 14 6 223 III P G/G A/A 0 3

10 58 F 11 64 14 4.8 242 III P+E G/G A/A 0 3

11 41 F 6 34 12 4 392 II P G/A G/A 2 3

12 64 F 6 46 10 4 393 III P+E G/A A/A 1 3

13 24 M 9 50 9 5.3 660 II P+E G/A A/A 1 6.25

14 37 M 6 48 8 4.9 399 II P+E A/A G/A 3 6.25

15 35 F 16 72 15 2.2 465 II P G/G G/A 1 6.25

16 62 F 8 35 8 4.9 525 III P+E G/G G/A 1 6.25¹17 26 F 10 55 8 7.9 465 III P G/G A/A 0 12.5

18 38 F 10 46 13 3 418 III P+E G/G G/A 1 12.5

19 41 F 7 68 8 3 538 II P+E G/G G/A 1 12.5

20 21 M 14 40 11 4 412 II P+E +Pros G/A A/A 1 12.5