Therapeutic Targeting of SDHB-Mutated Pheochromocytoma

Author Manuscript Published OnlineFirst on March 9, 2020; DOI: 10.1158/1078-0432.CCR-19-2335 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Therapeutic targeting of SDHB-mutated pheochromocytoma/paraganglioma with 2 pharmacologic ascorbic acid 3 4 Yang Liu1*, Ying Pang2*, Boqun Zhu2,3, Ondrej Uher2,4, Veronika Caisova2, Thanh-Truc 5 Huynh2, David Taieb5, Katerina Hadrava Vanova6, Hans Kumar Ghayee7, Jiri Neuzil4,8, Mark 6 Levine9, Chunzhang Yang1#, and Karel Pacak2# 7 8 Author affiliation: 9 1Neuro-Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, 10 Bethesda, Maryland 20892, USA 11 2Section on Medical Neuroendocrinology, Eunice Kennedy Shriver National Institute of Child 12 Health and Human Development, NIH, Bethesda, Maryland 20892, USA 13 3Endoscopy Center and Endoscopy Research Institute, Zhongshan Hospital, Fudan University, 14 Shanghai, 200032, PR China 15 4Department of Medical Biology, Faculty of Science, University of South Bohemia, Ceske 16 Budejovice, 370 05 Czech Republic 17 5Department of Nuclear Medicine, La Timone University Hospital, CERIMED, Aix-Marseille 18 University, 13385 Marseille, France 19 6Molecular Therapy Group, Institute of Biotechnology, Czech Academy of Sciences, Prague- 20 West, 252 50 Czech Republic 21 7Department of Internal Medicine, Division of Endocrinology, University of Florida College of 22 Medicine and Malcom Randall VA Medical Center, Gainesville, FL 32608, USA 23 8Mitochondria, Apoptosis and Cancer Research Group, School of Medical Science and Menzies 24 Health Institute Queensland, Griffith University, Southport, Queensland 4222, Australia 25 9Molecular and Clinical Nutrition Section, Intramural Research Program, National Institute of 26 Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA 27 28 29 *#Equal Contribution 30 31 Running title: Targeting ROS/labile iron overload pathway to treat SDHB-mutant PCPGs 32 33 Keywords: Pheochromocytoma/paraganglioma, SDHB mutations, reactive oxygen species, 34 ascorbic acid 35 36 Word count: 5,307 37 Number of figures: 6 and 3 supplementary 38 Number of tables: 2 supplementary 39 40 41 Corresponding author and person to whom reprint requests should be addressed: 42 43 Karel Pacak, M.D., Ph.D., D.Sc., FACE 44 Senior Investigator 45 Chief, Section on Clinical Neuroendocrinology

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1 Head, Developmental Endocrinology, Metabolism, Genetics and Endocrine 2 Oncology Affinity Group 3 Professor of Medicine 4 Section on Medical Neuroendocrinology 5 Eunice Kennedy Shriver National Institute of Child Health and Human Development 6 Building 10, Room 1-3140, MSC 1109 7 10 Center Drive, Bethesda, MD 20892 8 Tel: (301) 402-4594 9 Fax: (301) 402-4712 10 e-mail: [email protected] 11 12 13 Chunzhang Yang, Ph.D. 14 Investigator 15 Neuro-Oncology Branch Center for Cancer Research, NCI, NIH 16 Building 37, Room 1142E Bethesda, MD 20892 17 Phone: +1-240-760-7083 18 e-mail: [email protected] 19 20 Disclosure Statement: The authors declare no potential conflicts of interest. 21

1 Statement of translational relevance: Readership should be interested in this paper

2 because it demonstrates that SDHB-low Cluster I PCPG cells exhibited pseudohypoxia,

3 which reprogramed iron homeostasis and consequently resulted in high ROS generation.

4 This effect was enhanced by pharmacologic concentrations of ascorbic acid, presenting a

5 new approach for treating these types of cancers. Ascorbic acid is currently a treatment

6 agent in several clinical trials for acute myeloid leukemia, metastatic prostate cancer,

7 metastatic colon cancer, advanced non-small-cell lung cancer, and glioblastoma. Because

8 pharmacologic ascorbic acid has an excellent clinical safety profile, it is likely that ascorbic

9 acid can be a valuable tool for the treatment of PCPGs as well, for which few therapeutic

10 options exist.

1 Abstract

2 Purpose: Pheochromocytomas and paragangliomas (PCPGs) are usually benign

3 neuroendocrine tumors. However, PCPGs with mutations in the succinate dehydrogenase B

4 subunit (SDHB) have a poor prognosis and frequently develop metastatic lesions. SDHB-

5 mutated PCPGs exhibit dysregulation in oxygen metabolic pathways, including

6 pseudohypoxia and formation of reactive oxygen species (ROS), suggesting that targeting

7 the redox balance pathway could be a potential therapeutic approach.

8 Experimental design: We studied the genetic alterations of Cluster I PCPGs compared to

9 Cluster II PCPGs, which usually present as benign tumors. By targeting the signature

10 molecular pathway, we investigated the therapeutic effect of ascorbic acid on PCPGs using

11 in vitro and in vivo models.

12 Results: By investigating PCPG cells with low SDHB levels, we show that pseudohypoxia

13 resulted in elevated expression of iron transport proteins, including transferrin (TF),

14 transferrin receptor 2 (TFR2) and the divalent metal transporter 1 (SLC11A2; DMT1),

15 leading to iron accumulation. This iron overload contributed to elevated oxidative stress.

16 Ascorbic acid at pharmacologic concentrations disrupted redox homeostasis, inducing DNA

17 oxidative damage, and cell apoptosis in PCPG cells with low SDHB levels. Besides, through a

18 preclinical animal model with PCPG allografts, we demonstrated that pharmacologic

19 ascorbic acid suppressed SDHB-low metastatic lesions and prolonged overall survival.

20 Conclusions: The data here demonstrate that targeting redox homeostasis as a cancer

21 vulnerability with pharmacologic ascorbic acid is a promising therapeutic strategy for

22 SDHB-mutated PCPGs.

1 Introduction

2 Pheochromocytomas and paragangliomas (PCPGs) are catecholamine-producing

3 tumors, which are stratified into two major molecular subtypes: cluster I and cluster II.

4 Cluster I PCPGs, commonly exhibit abnormal activation of hypoxia signaling, particularly

5 those carrying mutations in succinate dehydrogenase subunits (SDHx), von Hippel-Lindau

6 (VHL), hypoxia-inducible factor 2A (HIF2A) or fumarate hydratase (FH). Cluster II PCPGs

7 commonly show hyperactivation of protein kinase pathways, carry mutations in ret proto-

8 oncogene (RET), neurofibromatosis type 1 (NF1), MYC associated factor X (MAX),

9 transmembrane protein 127 (TMEM127) or the kinesin family member 1B (KIF1B) (1-3).

10 Succinate dehydrogenase (SDH), also known as the mitochondrial respiratory complex II,

11 comprises SDHA, SDHB, SDHC, and SDHD subunits (4). Loss-of-function mutations in SDHx

12 lead to substantial loss of the complex II activity and results in reprogramming of cellular

13 metabolic pathways, as indicated by the accumulation of succinate, genome-wide

14 hypermethylation and a pseudohypoxia phenotype (5-10). Further, compromised complex

15 II disrupts electron transfer to oxygen, leading to increased formation of reactive oxygen

16 species (ROS) and redox imbalance (11,12).

17 Clinically, SDHB deficient PCPGs are one of the most common subtypes of Cluster I

18 PCPGs. In contrast to other PCPGs molecular subtypes, SDHB deficient PCPGs, especially

19 those producing and secreting catecholamines/metanephrines, typically present an early

20 onset and display tumor aggressiveness with development of metastases, all resulting in

21 patient mortality (13-16). For advanced PCPGs with metastatic lesions, surgical resection

22 coupled to radio- or chemotherapies are considered the standards of care. For

23 chemotherapy, the most widely used regimen is a combination of cyclophosphamide,

1 vincristine, and dacarbazine (CVD regimen) (17). However, emerging evidence shows that

2 in some patients, the CVD regimen produces only transient effects with only minimally

3 prolonged overall patient survival (18). Therefore, therapeutic approaches with enhanced

4 efficacy have been advocated to better handle advanced PCPGs.

5 The pseudohypoxic ‘signature’ and angiogenic pathways have been proposed as

6 targets for limiting tumor expansion in Cluster I PCPGs (19). Humanized monoclonal

7 VEGFA antibody and tyrosine kinase inhibitors showed efficacy in Cluster I PCPGs and have

8 advanced to clinical studies (20,21). Similarly, disturbance of the NAD+/NADH metabolism

9 in Cluster I PCPGs has been reported, establishing a therapeutic vulnerability to PARP

10 inhibitors (22). Overall, the distinctive pattern in molecular signaling and metabolism in

11 Cluster I PGPCs suggests that there may be novel therapeutic targets for this type of

12 malignancy.

13 To expand the therapeutic possibilities for Cluster I PCPGs, we investigated the redox

14 homeostasis in SDHB deficient cells. Further, we hypothesized and probed the associations

15 between metabolic deficiency and iron uptake in these cells. Finally, we evaluated the

16 impact of pharmacologic ascorbic acid on Cluster I PCPGs using in vitro assays and an

17 animal model.

1 Materials and Methods

2 This study was approved by the Institutional Review Board of the Eunice Kennedy

3 Shriver NICHD/NIH, and all patients gave written informed consent.

5 Patient samples

6 All patient samples, including frozen tissue and Formalin-Fixed Paraffin-Embedded

7 (FFPE) slides, were collected from clinically identified Cluster I and Cluster II PCPG patients

8 (Supplementary Table S1). Tissue preparation and dissection were performed as

9 previously reported (22), and clinical samples used are summarized in the supplemental

10 table.

12 Cell lines and reagents

13 Mouse pheochromocytoma (MPC) cells (4/30 PRR) (23) were cultured in Dulbecco’s

14 modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS)

15 and penicillin/streptomycin at 37 °C. To establish the SDHB wildtype (SDHBWT) and SDHB

16 knockdown (SDHBKD) cell lines, MPC cells were transduced by lentivirus with either non-

17 targeted or SDHB-targeted short hairpin RNA (shRNA). The specific targeting sequencing

18 for SDHB was TGA GTA ACT TCT ACG CAC A (GE Dharmacon). The virus was added to the

19 supernatant of the cell culture medium and cultured at 37°C, 5% CO2, for three days. Then,

20 the cells were selected by puromycin (2 g/mL). Human pheochromocytoma precursor

21 cells (hPheo1) (24) were cultured in Roswell Park Memorial Institute (RPMI) 1640

22 medium with 10% FBS, and SDHB was knocked down using a lentivirus with shRNA

23 (targeting sequence: CAG AGC TGA ACA TAA TTT A; GE Dharmacon). Control hPheo1 cells

1 were transduced with non-targeted shRNA. Knockdown efficacy was evaluated in all

2 transformed cells by quantitative real-time PCR and western blot. For in vivo studies, SDHB

3 knockdown MPC cells were transduced with a retrovirus containing firefly luciferase and

4 selected using G418. Human renal carcinoma cells University of Michigan-Renal

5 Carcinoma-6 (UMRC6) (25,26) were cultured in Dulbecco’s modified Eagle’s medium

6 (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and

7 penicillin/streptomycin.

8 Ascorbic acid, N-acetyl-L-cysteine (NAC), and catalase (Sigma-Aldrich) were dissolved

9 in phosphate-buffered saline (PBS) before being added to the cell medium. The final

10 concentrations of ascorbic acid, NAC, and catalase were 1 mM, 2.5 mM, and 500 U/mL,

11 respectively. The pH of the medium was adjusted to 7.4 before use. Cells were treated

12 overnight for oxidative stress and DNA damage experiments and treated 24 h for

13 experiments related to cell viability.

15 ROS measurement

16 Cells were seeded in a 96-well plate and treated under various conditions. Cellular

17 levels of H2O2 were determined using the ROS-Glo™ H2O2 Assay kit (Promega). Briefly,

18 derivatized luciferin substrate from the kit was incubated with samples, reacting directly

19 with H₂O₂ to generate the luciferin precursor. After the addition of the ROS-Glo™ Detection

20 Solution, which converts the luciferin precursor to luciferin, the luminescence signal was

21 recorded by a FLUOstar Omega plate reader (BMG Labtech). The ROS luminescence signal

22 was normalized to protein quantification. Mitochondrial ROS levels were estimated by

23 MitoSOX staining (Thermo Fisher Scientific). Briefly, cells were incubated with 5 M

1 MitoSOX-Red for 30 min at 37 °C. The fluorescence signal was assessed on a FACSCanto II

2 flow cytometer (BD Biosciences), and fluorescence intensity was quantified using Image J

3 (version 1.52K, National Institutes of Health).

5 Cellular labile iron pool quantification and iron assay

6 The labile iron pool level was evaluated using Calcein red-orange AM fluorescent dye

7 (Thermo Fisher Scientific) combined with 2’,2’-bipyridyl (BIP), as previously described

8 (27). Briefly, 106 cells were pelleted and resuspended in 1 mL PBS with 500 nM Calcein

9 red-orange AM. After incubation for 15 min at 37 °C (5% CO2), cells were washed with PBS

10 and resuspended in 1 mL PBS. The cells were divided into two flow cytometry tubes, and

11 500 M BIP was added to one tube. Cells were then incubated at room temperature for

12 more than 15 min before analysis using an LSRFortessa flow cytometer (BD Biosciences).

13 The labile iron pool (A.U.) = MFIBIP – MFINoBIP and the relative labile iron pool for each

14 sample were compared to the control sample.

15 Iron was quantified with the Iron Assay Kit (Sigma-Aldrich). Cells or tissue specimens

16 were collected and rapidly homogenized in cold iron assay buffer from the kit. Ferrous and

17 total iron from samples were assessed following the manufacturer’s protocol, and

18 absorbance was taken at 593 nm using the FLUOstar Omega plate reader.

20 RNA interference

21 Small interference RNA was designed and purchased from Integrated DNA

22 Technologies (IDT). The sequences of small interference RNA oligos used in this study are

23 listed in Supplementary Table S2.

2 ARE Luciferase reporter assay

3 Nrf2-driven transcriptional activation was quantified as ARE transcriptional activity

4 using a dual-luciferase reporter system (28,29). pGL3.47-ARE-Luc plasmid (900 ng) and

5 100 ng pRL-TK plasmid (Promega) were co-transfected into 8,000 cells using

6 Lipofectamine 3000 (Thermo Fisher Scientific). The firefly (ARE activity) and renilla

7 (internal control) luminescence signal were assessed using a Polarstar Optima plate reader

8 (BMG LABTECH).

10 Cell viability analysis

11 The dose-response curve of ascorbic acid treatment was assessed with the CCK-8 assay

12 (Dojindo). Cells were seeded in a 96-well plate and treated with ascorbic acid for 24 h.

13 After replacing the culture medium with fresh medium, CCK8 was added, and the cells were

14 incubated at 37 ℃ for one h. The absorbance at 450 nm was assessed using a FLUOstar

15 Omega plate reader. Cells were enumerated using a Vi-CELL cell counter (Beckman Coulter,

16 Brea, CA) to evaluate cell viability after treatment.

18 Oxidative DNA damage ELISA assay

19 Oxidative DNA damage was assessed by the DNA/RNA Oxidative Damage ELISA kit

20 (Cayman Chemical) according to the manufacturer’s instructions. Briefly, DNA was purified

21 from cell lysates, digested with nuclease P1, and incubated with alkaline phosphatase for

22 30 min at 37 °C. Digested DNA samples were then incubated in the provided plate for 18 h

23 at 4 ℃. Next, the samples were incubated with freshly reconstituted Ellman’s reagent on an

1 orbital shaker for 90 min. The absorbance of each well was assessed on a FLUOstar Omega

2 plate reader at 405 nm.

4 Immunohistochemistry

5 Immunohistochemistry was performed as previously reported (22). Briefly, 8 µm

6 formalin-fixed, paraffin-embedded tissue sections from patients or animals were

7 deparaffinized in xylene three times and rehydrated in a series of ethanol solutions ranging

8 from 100 to 75%. The slides were boiled in sodium citrate buffer for 20 min for antigen

9 retrieval and blocked with blocking solution (Thermo Fisher Scientific) for 30 min at room

10 temperature. Next, slides were probed with anti-TF, anti-TFR2 (Thermo Fisher Scientific),

11 or anti-DMT1 (Abcam) antibodies overnight and developed with the DAB+ substrate

12 chromogen system (Dako). Slides were counterstained with hematoxylin, dehydrated, and

13 mounted. Samples were visualized using a KEYENCE microscope with 40-fold

14 magnification.

16 Alkaline comet assay

17 An alkaline comet assay was performed as previously described (22,30) to analyze

18 DNA damage levels. Briefly, cells were harvested and resuspended in molten agarose and

19 evenly spread on pre-coated glass slides. Cells on the slides were lysed and subjected to

20 electrophoresis for 30 min at 1 V/cm. Slides were stained with diluted SYBR Safe

21 (Invitrogen) and visualized by fluorescence microscopy (KEYENCE).

23 DNA fragmentation assay

1 Genomic DNA was collected and purified with the DNeasy Blood & Tissue Kit (Qiagen)

2 according to the manufacturer’s protocol. Five hundred DNA samples were resolved by

3 electrophoresis in 4-20% Novex TBE gel (Thermo Fisher Scientific). The gel was stained

4 with SYBR Safe DNA Gel Dye for 20 min and visualized in a ChemiDoc Imaging System (Bio-

5 Rad).

6 Mitochondria were isolated from each sample, and a mitochondrial DNA damage assay

7 was performed as previously described (31). The long (10.1 kb) and short (241 bp)

8 fragments from the mitochondrial genome were amplified by semiquantitative PCR with

9 the primers are listed in Supplementary Table S2. The PCR products were resolved in a 1%

10 agarose gel, stained with SYBR Safe DNA Gel Dye, and visualized in a ChemiDoc Imaging

11 system.

13 Annexin V/PI apoptosis assay

14 Cell apoptosis was measured using Annexin V/PI staining (Thermo Fisher Scientific).

15 Cells (including the culture media) were harvested and washed once with PBS and then

16 incubated with Annexin V and PI for 20 min on ice. The fluorescence signal was measured

17 on a FACSCanto II flow cytometer, and the percentage of apoptotic cells was quantified.

19 Caspase 3/7 activity assay

20 The activity of caspase 3/7 was determined using the Caspase-Glo 3/7 assay kit

21 (Promega). In brief, cells were seeded in a 96-well plate and incubated with the Caspase-

22 Glo 3/7 reagent for 30 min. The luminescence signal was evaluated using a Polarstar

23 Optima plate reader.

2 Western blotting

3 Cells or tissue samples were collected, and total protein was extracted in ice-cold RIPA

4 lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (Thermo

5 Fisher Scientific). Total protein (20-30 µg) was resolved using the NuPAGE 4-12% Novex

6 Bis-Tris Gel (Invitrogen). The proteins were transferred to PVDF membranes (Bio-Rad) and

7 probed with primary antibodies at 4℃ overnight. The membrane was then washed with

8 PBST (PBS+0.1% Tween20), incubated with HRP-conjugated secondary antibodies, and

9 visualized using a ChemiDoc Imaging System. Anti--actin IgG (Cell Signaling Technology,

10 1:5,000) was used as an internal control. The primary antibodies used included anti-HIF-

11 1, anti-HIF-2, anti-DMT1 (Abcam), anti-TF, and anti-TFR2 (Thermo Fisher Scientific). All

12 primary antibodies were diluted 1:1,000.

14 Quantitative real-time PCR

15 Cell or tissue samples were dissected, and total RNA was extracted using the RNeasy

16 Mini kit (Qiagen). RNA (1 g) was reverse transcribed to cDNA using the SuperScript III

17 First-Strand Synthesis System (Invitrogen). Primer sets for each gene are listed in

18 Supplementary Table S2.

20 Chromatin immunoprecipitation (ChIP) assay

21 Cells were seeded in 150 mm Petri dishes, and a ChIP assay was performed using the

22 ChIP-IT High Sensitivity Kit (Active Motif) according to the manufacturer’s protocol. Cells

23 (1.5 x 107) were fixed, and chromatin was prepared from each sample. Thirty micrograms

1 of the chromatin preparation were fragmented by sonication and precipitated with anti-

2 HIF-1 antibody (Abcam). Purified ChIP DNA was analyzed by quantitative real-time PCR

3 to measure promoter enrichment, comparing pull-down DNA and input DNA. The

4 sequences of the primer set used are listed in Supplementary Table S2.

6 Cell viability measured by trypan blue exclusion assay

7 One hundred thousand cells were seeded in a 6-well plate. To determine viability, cells

8 were detached with 0.05% trypsin. All the culture media and wash buffer were collected

9 for the analysis. The measurement of cell viability was assessed by a Vi-Cell XR cell counter

10 (Beckman Coulter) using a trypan blue exclusion staining protocol. The cell suspension was

11 mixed with an equal volume of trypan blue. Fifty random images were taken to determine

12 the live and dead cells.

14 Allograft animal model

15 Animal experiments were performed following the principles and procedures of the

16 Eunice Kennedy Shriver NICHD animal protocol (ASP 15-028) approved by the Animal Care

17 and Use Committee of the National Institutes of Health. Eight-week-old female athymic

18 mice (Jackson Laboratory) were injected in the tail vein with 1.5 x 106 MPC-SDHBKD-Luci or

19 MPC-SDHBWT-Luci cells. Tumor growth was assessed on an IVIS in vivo imaging system

20 every week. Two weeks after the tail vein injection, the luminescence signals could be

21 detected in the liver or spleen, and then mice were randomly divided into two groups (10

22 mice/group for SDHBWT cells, 10 mice/group for SDHBKD cells) and treated with saline

23 solution or ascorbic acid. Ascorbic acid was dissolved in saline solution and neutralized to

1 pH 7.3 with sodium hydroxide. The freshly prepared ascorbic acid solution was injected

2 intraperitoneally every day at 4 g/kg (32,33). The behavior and body weight of animals

3 was monitored every day. Animals were sacrificed, and metastatic lesions in the liver were

4 harvested at the end for further evaluation.

6 Colony formation

7 SDHBWT and SDHBKD MPC cells were seeded in a 6-well plate at a density of 2,000 cells per

8 well. Cells were treated with 0.5 mM ascorbic acid for two weeks. Then, the cells were fixed

9 in 4% cold formaldehyde (Sigma) and stained with 2% crystal violet (Sigma). This

10 experiment was repeated by three replicates, and the images were quantified by ImageJ.

12 Statistical analysis

13 The statistical significance of differences between the two groups was analyzed using

14 the Student’s t-test. All statistical tests were two-sided. The results were presented as mean

15 value ± standard error of the mean (SEM). A P value of less than 0.05 was considered

16 statistically significant. The statistical tests in the current study were analyzed using

17 GraphPad Prism 7.01 (GraphPad Software).

1 Results

2 SDHB deficiency triggers pseudohypoxia and reprograms iron metabolism

3 Genetic defects in SDHx result in substantial loss of mitochondrial complex II (SDH and

4 succinate-ubiquinone oxidoreductase activity) activity, leading to abnormal activation of

5 the hypoxia signaling pathway and a switch to the pseudohypoxia phenotype (10). To

6 investigate the impact of pseudohypoxia in Cluster I PCPGs, we established cell lines with

7 considerably decreased levels of SDHB using MPC and hPheo1 cells. Knockdown efficiency

8 was confirmed by western blotting (Figure 1A and Supplementary Figure 1A), and

9 quantitative real-time PCR (Figure 1B). We found that decreased levels of SDHB led to

10 substantial activation of the hypoxia signaling pathway resulting in upregulation of genes

11 that regulate iron homeostasis, such as transferrin (TF), transferrin receptor 2 (TFR2) and

12 solute carrier family 11 member 2 (SLC11A2, DMT1; Figure 1C and D and Supplementary

13 Figure 1B). We confirmed activation of the hypoxia signaling pathway, as well as the

14 upregulation of genes that are associated with iron metabolism in Cluster I PCPGs tumor

15 specimens obtained from patients (Figure 1E, F, and G). To further validate the correlation

16 between the hypoxia signaling pathway and iron homeostasis in an SDHB-low background,

17 we performed chromatin immunoprecipitation (ChIP) assay. This assay revealed stronger

18 affinity of HIF-1 to the promoter regions of TF, TFR2, and SLC11A2 in the SDHB

19 knockdown (SDHBKD) cells compared to their wild type counterparts, indicating that HIF-

20 1 mediated transcriptional activation of genes that regulate iron transport (Figure 1H and

21 Supplementary Figure 1C). Consistent with the up-regulation of iron transporters, we

22 found elevated intracellular labile iron in SDHBKD cells compared with SDHBWT cells (Figure

23 1I, Supplementary Figure 1D, and 1E). Re-expression of SDHB reduced labile iron to

1 baseline level in SDHBKD cells (Supplementary Figure 1F). Genetic silencing of TF, HIF-1, or

2 HIF-2 by small interfering RNA reduced the iron level in SDHBKD cells (Figure 1J, 1K, 1L,

3 and Supplementary Figure 1G). Besides, we measured the expression of TF and SLC11A2

4 under chemical hypoxic conditions. Both SDHBWT and SDHBKD cells showed strong

5 induction of TF and SLC11A2 expression under cobalt chloride treatment (Supplementary

6 Figure 1H). Moreover, the expression of TF and TFR2 was measured in a VHL-low UMRC6

7 cell line. Results showed that the re-expression of VHL in UMRC6 cells led to reduced

8 expression of TF and TFR2 and reduced labile iron pools (Supplementary Figure 1I and 1J).

9 These findings suggest that activation of the hypoxia (pseudohypoxia) signaling pathway in

10 SDHBKD cells resulted in TF-dependent ferrous iron overload.

12 Iron overload compromises redox balance in SDHBKD cells

13 Previous findings indicated that abnormal iron metabolism results in oxidative stress

14 in a variety of malignancies, including SDHB-deficient ones (34,35). Using the ROS-Glo

15 assay, we showed that a decrease of SDHB correlated with a significant elevation of

16 oxidative stress (Figure 2A and Supplementary Figure 1K). A MitoSOX assay confirmed

17 increased ROS in the mitochondria of SDHBKD cells (Figure 2B and Supplementary Figure

18 1L). Moreover, the antioxidant response element (ARE) luciferase reporter assay revealed

19 that SDHBKD cells exhibited higher ARE-dependent transcriptional activity, indicating

20 increased oxidative stress (Figure 2C). Iron overload plays a crucial role in deleterious

21 consequences of elevated ROS through the Fenton reaction, which converts H2O2 into

22 hydroxyl radicals via an iron-dependent mechanism (36). We confirmed that the iron

23 overload in SDHBKD cells was correlated with elevated oxidative stress, as increased ROS

1 levels were suppressed in the presence of the exogenous iron chelator deferoxamine (DFO).

2 In SDHBKD cells, ROS was suppressed by DFO, consistent with the notion that an elevated

3 iron pool in these cells mediates ROS formation (Figure 2D). Similarly, RNA interference

4 targeting TF lowered the level of ROS, suggesting that the internalization of iron could

5 promote the enhancement of oxidative stress (Figure 2E). Also, incubation of MPC cells in

6 high iron media with 500 M ferric chloride or 500 M ferrous chloride for 18 hr resulted

7 in increased levels of ROS and higher cytotoxicity, confirming the association between iron

8 overload and disrupted redox homeostasis (Figure 2F, 2G, and Supplementary Figure 1M).

10 Ascorbic acid induces enhanced cytotoxicity in SDHB deficient cells

11 Pharmacologic concentrations of ascorbic acid (>1 mM) result in cytotoxicity in cancer

12 cells via the generation of H2O2 (32,37). We hypothesized that pharmacologic

13 concentrations of ascorbic acid could serve as an anti-tumor agent for SDHB mutant PCPGs

14 via synergism with the iron overload found in SDHBKD cells. Findings were that, in SDHBKD

15 cells, ascorbic acid further enhanced ROS levels, which were reversed by NAC or catalase

16 (Figure 3A). Accumulation of ROS was also reduced by the re-expression of wild type SDHB

17 (Supplementary Figure 1N). Consistently, both the ARE luciferase reporter assay and the

18 MitoSOX Red assay showed increased ROS after ascorbic acid treatment in both cell lines,

19 and higher ROS levels in SDHBKD compared to SDHBWT cells (Figure 3B-D, Supplementary

20 Figure 1K, and 1O). The reduction of ROS levels upon catalase addition indicates that at

21 least some effects of pharmacologic ascorbic acid require the formation of extracellular

22 hydrogen peroxide (37-39). To further probe whether iron metabolism underlies redox

23 imbalance, we supplemented SDHBWT and SDHBKD cells with exogenous iron and ascorbic

1 acid and found that iron enhanced ROS with ascorbic acid (Figure 3E). Moreover, we found

2 elevated labile iron in both SDHBWT and SDHBKD cells after ascorbic acid treatment,

3 although labile iron in SDHBKD cells was higher than in wildtype cells, and knockdown of TF

4 reduced labile iron (Supplementary Figure 2A). DFO treatment significantly reduced the

5 elevation of labile iron (Supplementary Figure 2B). However, DFO did not reverse

6 ascorbate-induced oxidative DNA damage (Supplementary Figure 2C and 2D), suggesting

7 additional mechanisms of pharmacologic ascorbate action distinct from those related to

8 iron-associated metabolism.

9 To further evaluate whether ascorbic acid is a therapeutic candidate for the treatment

10 of cancers with mutated SDHB, we characterized concentration-dependent cytotoxicity.

11 SDHB deficiency predisposed cells to be sensitive to ascorbic acid-induced cytotoxicity,

WT 12 with IC50 = 1.859 mM (36 pmol/cell) for SDHB cells and IC50 = 0.330 mM (6 pmol/cell)

13 for SDHBKD cells (Figure 4A). Further, we found that ascorbic acid enhanced oxidative

14 damage in SDHBKD cells, as shown by 8-OH-dG ELISA and mitochondrial DNA oxidative

15 damage assays (Figure 4B and 4C, Supplementary Figure 2E). Accordingly, DNA

16 electrophoresis and the comet assay revealed increased DNA fragmentation in SDHBKD cells

17 treated with ascorbic acid compared to SDHBWT cells (Figure 4D-4F, Supplementary Figure

18 2F). The DNA fragmentation was prevented with catalase, consistent with the formation of

19 hydrogen peroxide as a prerequisite for and initiator of subsequent DNA damage. The

20 ascorbic acid-induced DNA damage was associated with the onset of the apoptotic cascade,

21 as evidenced by the caspase-3/7, annexin V-PI cell apoptosis assay, and CCK8 cell viability

22 assay (Figure 4G-J). Furthermore, the clonogenic assay showed that ascorbic acid

23 significantly reduced colony formation of SDHBKD MPC cells (Figure 4K). Moreover, the

1 trypan blue exclusion assay showed reduced cell number of SDHBKD cells compared with

2 SDHBWT counterparts (Figure 4L), while re-expression of SDHB reversed ascorbic acid-

3 induced cytotoxicity (Supplementary Figure 2G). ROS scavengers, such as NAC or catalase,

4 largely reversed cytotoxicity induced by ascorbic acid, again suggesting that the

5 genotoxicity is ROS-dependent.

7 Ascorbic acid suppresses SDHBKD metastatic lesions and improves disease outcome

8 To explore the effects of pharmacologic ascorbic acid administration on SDHB

9 interference in vivo, we established metastatic pheochromocytoma tumors by injecting

10 SDHBWT or SDHBKD MPC cells into athymic nude mice (22). Bioluminescence imaging

11 revealed that pharmacologic ascorbic acid (4g/kg/day) strongly delayed the expansion of

12 the metastatic lesions in the SDHBKD group. Four weeks after treatment, tumor growth was

13 suppressed by approximately 80% in mice with SDHBKD allografts after receiving ascorbic

14 acid (Figure 5A, 5B, and Supplementary Figure 3). Importantly, ascorbic acid improved

15 disease outcome of the SDHBKD allograft-bearing mice, with median survival increased by

16 21.8% (median survival: saline, t = 27.5 d; ascorbic acid, t = 33.5 d; p = 0.0014, Figure 5C).

17 For further validation, we assessed oxidative damage by measurement of 8-OH-dG in

18 metastatic lesions dissected from the liver. In SDHBKD allograft tissues, increased oxidative

19 damage was found in hepatic lesions from the ascorbic acid-treated mice compared to the

20 saline group (Figure 5D). Moreover, the histologic analysis showed higher Ki67 expression

21 in the saline group and increased H2A.X expression in the ascorbic acid treatment group

22 (Figure 5E-5F), indicating elevated DNA damage and cytotoxicity. In the SDHBWT group,

23 ascorbic acid treatment alone did not significantly inhibit tumor growth nor prolong

1 survival. (Figure 5A-5C, Supplementary Figure 6). No noticeable weight loss or other side

2 effects were observed in the experimental animals.

1 Discussion

2 In the present study, we demonstrated that Cluster I PCPGs with SDHB deficiency

3 exhibited increased expression of iron transport genes, which were induced by activation

4 of the transcriptional factor HIF-α. Increased intracellular iron transport contributed to an

5 increase in the labile iron pool and elevated ROS burden in SDHBKD cells. Pharmacologic

6 levels of ascorbic acid aggravated the oxidative burden of SDHB deficient Cluster I PCPGs,

7 leading to genetic instability and apoptotic cell death. Our study highlights the importance

8 of the labile iron pool and ROS balance in Cluster I PCPGs and reveals that targeting the

9 intrinsic redox status could be a potential therapeutic strategy for these tumors (Figure 6).

11 Pseudohypoxia triggers iron overload in SDHBKD cells

12 Transcriptomic profiling revealed that Cluster I PCPGs exhibit signatures of hypoxia-

13 related gene expression, suggesting activation of HIF- and increased expression of its

14 downstream genes that regulate angiogenesis, tumor growth, and energy metabolism (40-

15 42). Here, we demonstrate that SDHBKD cells recapitulate the pseudohypoxia phenotype

16 observed in human tumors, evidenced by the upregulation of hypoxia-related genes, such

17 as TF, TFR2, SLC11A2, EPO, PGK1, and LDHA. Importantly, TF, TFR2, and DMT1 are critical

18 components of cellular iron uptake, and the upregulation of these proteins indicates an

19 increased level of iron in SDHBKD cells. It has been reported that as a major iron-binding

20 and -transporting protein, increased level of TF could lead to ferric iron elevation, which

21 could induce ferroptosis, an iron-dependent type of cell death (43). Conversely, a missense

22 mutation at a splice junction of SLC11A2 that impaired iron uptake in erythroid cells and

23 led to iron deficiency-related anemia was discovered in a patient (44). Another study

1 described the overexpression of the DMT1 and TFR proteins in human colonic carcinoma,

2 resulting in increased intracellular iron by means of increased intracellular iron import and

3 reduced iron efflux (45). Besides pseudohypoxia-induced iron uptake, SDHB deficiency

4 results in loss of mitochondrial Fe-S cluster, which contributes to the labile iron elevation

5 and vulnerability to oxidative damage (46). Our data further demonstrate that increased

6 expression of iron transport proteins is a critical component of iron overload-induced by

7 pseudohypoxia in SDHB-disrupted tumor cells.

9 Iron overload: The Sword of Damocles for SDHB deficient malignancies

10 As an important part of DNA synthesis, cellular respiration, and redox balance, iron

11 plays a crucial role in cell metabolism and proliferation (47). However, because of the

12 harmful effect of an overload of “free” iron, the labile iron pool is usually maintained at the

13 lowest sufficient level by tight regulation of the expression of proteins that are associated

14 with iron transport and homeostasis (34). High levels in the labile iron pool are often

15 observed during malignant transformation, together with the disruption of cellular redox

16 homeostasis (27). Redox-active iron converts hydrogen peroxide largely to the highly toxic

2+ 3+ 17 hydroxyl radical via the Fenton/Haber–Weiss reaction cycles (Fe + H2O2 → Fe + HO• +

3+ 2+ KD 18 OH−; Fe + H2O2 → Fe + HOO• + H+) (48). Thus, elevated labile iron pool in SDHB cells

19 could prime cancer cells for ascorbic acid-induced oxidative damage. In the current study,

20 we found high levels of cellular and mitochondrial ROS in SDHBKD cells and depletion of

21 iron by using DFO reduced oxidative stress in SDHBKD cells, suggesting that ROS

22 accumulation was due to the presence of the intracellular labile iron pool. Genetic silencing

23 of TF reduced the labile iron pool as well as the ROS level indicating a close association

1 between low levels of SDHB, iron overload, and oxidative stress, consistent with reports

2 that showed a correlation between ROS accumulation and SDH dysfunction (49,50). One

3 study based on prokaryotic SDH suggested that electron transport chain dysfunction

4 induced by SDH deficiency was associated with ROS formation (51). Moreover, blocking

5 interception of electrons generated at SDHA by displacement of their acceptor, coenzyme Q,

6 from the membrane part of CII by vitamin E succinate and, even more so, its mitochondria-

7 targeted variant caused the massive formation of ROS with ensuing apoptosis induction

8 (52). Our findings show that the increased labile iron pool induced by pseudohypoxia in

9 SDHBKD cells contributes to ROS generation.

10 Besides iron overload and oxidative cellular damage, other ascorbic acid-associated

11 molecular mechanisms could be relevant for tumor suppression. SDH-depletion leads to

12 the accumulation of succinate, which serves as a competitive inhibitor of DNA/histone

13 demethylases (53). Inhibition of the demethylation reactions s eventually leads to a

14 genome-wide hypermethylation phenotype in these cancer cells (54). Conversely, ascorbic

15 acid has been shown to remodel the epigenome by enhancing histone demethylases and

16 ten-eleven translocation (TET) protein (55,56). Using TET as an example, ascorbic acid

17 reduces Fe3+ to Fe2+, which then fuels the demethylation steps from 5-methylcytosine

18 (5mC) to 5-hydroxymethylcytosine (5hmC), 5hmC to 5-fluorocytosine (5fC), and 5fC to 5-

19 carboxylcytosine (5caC), respectively (39,57). Thus, it is possible that the presence of high

20 concentrations of ascorbic acid partially rectifies the hypermethylation phenotype, and

21 therefore limits the aggressive phenotypes during cancer growth, here particularly SDHB-

22 PCPG. Investigation of epigenome reprogramming in SDH-depleted cancers will be of

23 importance to elucidate the molecular mechanisms of ascorbic acid treatment.

2 Ascorbic acid as a potential therapy for cancer

3 Ascorbic acid has been shown to act as an anti-cancer agent for certain advanced

4 tumors, such as pancreatic and ovarian cancer (58,59). A single-arm pilot trial also

5 revealed that about half of the patients with refractory acute myeloid leukemia or

6 myelodysplastic syndromes exhibited a clinical response to ascorbic acid administration

7 (60). Studies on several types of tumor cells with increased steady-state ROS levels, due to

8 oxidative metabolism defects, and with an increased labile iron pool (61) revealed that

9 pharmacologic doses of ascorbic acid could be used as a potential therapeutic agent by

10 targeting oxidative metabolic pathways (27). It has been suggested that iron-enriched

11 tumors, such as non-Hodgkin’s lymphomas and renal cell carcinomas, are also good

12 candidates for ascorbic acid trials (38). In our present study, ascorbic acid increased ROS to

13 higher levels in SDHBKD cells that had increased levels of iron, indicating ROS-mediated

14 toxicity of ascorbic acid for SDHB PCPGs.

15 While ascorbic acid at pharmacologic concentrations has few side effects in healthy

16 organs or tissues, it is more toxic to tumor cells with higher pro-oxidative status (27). Two

17 clinical trial data sets have determined that intravenous administration of ascorbic acid,

18 was e well tolerated and showed efficacy in advanced and metastatic diseases (33,59). In

19 healthy tissues, ROS and labile iron pool levels are maintained at a basal level, increased

20 ROS induced by the labile iron pool plus pharmacologic ascorbic acid are rapidly

21 neutralized by elevated removal of hydrogen peroxide or by normal cellular repair

22 mechanisms (39). However, for tumors with higher labile iron pool and ROS levels, it is

23 difficult to eliminate the severely increased ROS induced by ascorbic acid (27).

1 SDHB deficient PCPGs are examples of tumor types sensitive to pharmacologic doses of

2 ascorbic acid. Ascorbic acid induces more oxidative DNA damage and efficiently promotes

3 apoptosis in SDHBKD cells, which exhibit a higher ROS stress burden than their SDHBWT

4 counterparts with lower ROS and labile iron pool levels (Figure 4). Furthermore, ROS

5 scavengers, such as NAC and catalase, reverse the genotoxicity and cytotoxicity in these

6 cells, strongly indicating the central role of ROS in ascorbic acid-induced cell death and of

7 an increased labile iron pool as a critical modulator of elevated ROS generation. Aberrant

8 iron metabolism combined with redox disbalance induced by ascorbic acid, could be a

9 promising therapeutic target for malignancies, especially those with ‘fragile’ redox

10 homeostasis. This approach is particularly appealing, given the safety and low adverse

11 effects of pharmacologic doses of ascorbic acid. Nevertheless, our study shows that the

12 SDHBKD allograft growth was delayed but not eliminated by pharmacological ascorbic acid.

13 One of our recent findings showed that the activity of nuclear factor erythroid 2-related

14 factor 2 (NRF2) is significantly up-regulated in SDH-depleted cells. In accordance with this,

15 many antioxidant genes, such as HMOX1, GCLC, GCLM, and SLC7A11, are highly presented in

16 this disease subtype (62). It is possible that in SDH-depleted cancers, the intrinsic

17 antioxidant pathway is up-regulated, which partially counteracts ascorbic acid-induced

18 oxidative damage. Combination treatment with an NRF2/antioxidant pathway inhibitor

19 may be useful to synergize with ascorbic acid and achieve a better disease outcome. SDHB-

20 depleted tumors are generally resistant to standard chemotherapy. Ascorbate here was

21 used as a single treatment agent, both for wildtype and mutant tumors in athymic mice.

22 Other cell and animal studies with highly aggressive and treatment-resistant cancer cell

23 types, i.e., pancreatic, ovarian, glioblastoma, show that ascorbate as a single treatment

1 agent has some but not full efficacy (32,63). A combination of pharmacologic ascorbate

2 with other agents has often reported being synergistic (37,63), including a recent report

3 using combination treatment with pharmacologic ascorbate plus anti-PD1agent (64).

5 In summary, we demonstrate that Cluster I SDHB-low PCPG cell lines and SDHB

6 deficient tumors feature an increased labile iron pool induced by the HIF signaling pathway,

7 leading to higher levels of ROS. Disruption of this redox state by ascorbic acid may cause an

8 overwhelming ROS burden, which presents a potential strategy toward this tumor type

9 treatment. The difficult-to-treat PCPGs are typified by a frequency of nonsense SDHB

10 mutations showing a proof-of-concept for this approach as well as candidates for clinical

11 trials.

1 Figure legends

2 Figure 1. SDHB deficiency induces pseudohypoxia and reprograms iron homeostasis.

3 A. SDHB knockdown efficiency in MPC and hPheo1 cell lines was measured by western

4 blot (upper panel) and quantification (lower panel, n=3). -actin was used as a

5 loading control.

6 B. SDHB knockdown efficiency in MPC and hPheo1 cell lines was measured by

7 quantitative real-time PCR (n=4).

8 C. HIF-1, HIF-2, TF, TFR2, and DMT1 proteins in SDHBWT and SDHBKD MPC cells

9 were measured by western blot (left panel) and quantification (right panel, n=3). -

10 actin was used as a loading control.

11 D. Levels of TF, TFR2, SLC11A2 (DMT1), EPO, PGK1, and LDHA mRNA were measured

12 by quantitative real-time PCR. Data normalized to SDHBWT.

13 E. Levels of HIF1A, HIF2A, TF, TFR2, SLC11A2, EPO, TFRC, and LDHA mRNA in Cluster I

14 and Cluster II PCPGs Frozen tissues were measured by quantitative real-time PCR.

15 CI, Cluster I (n=5-8); CII, Cluster II (n=4-7).

16 F. Expression of DMT1, TF, and TFR2 in Cluster I (n=4) and Cluster II (n=5) PCPGs

17 were measured by immunohistochemistry staining obtained from Cluster I and

18 Cluster II PCPGs FFPE tissues. Bar = 50 m. NAM, normal adrenal medulla; CI,

19 Cluster I; CII, Cluster II.

20 G. Integrated optical density quantification of the immunohistochemistry staining

21 shown in Figure 1F. Cluster I (CI), n=4; Cluster II (CII), n=5.

22 H. Promoter affinity of HIF-1 assay in SDHBWT and SDHBKD hPheo1 cells was

23 measured by a ChIP PCR assay.

1 I. The level of the labile iron pools in SDHBWT and SDHBKD MPC cells was measured by

2 a cellular labile iron pool quantification assay.

3 J. Knockdown efficiency of siRNA targeting TF in MPC cells was measured by western

4 blot (upper panel) and quantitative real-time PCR (lower panel).

5 K. The level of the labile iron pools in SDHBWT and SDHBKD MPC cells after TF

6 knockdown was measured by a cellular labile iron pool quantification assay.

7 L. The level of the labile iron pools in SDHBKD MPC cells after HIF1A and HIF2A

8 knockdown was measured by a cellular labile iron pool quantification assay. All

9 significance in differences was determined by t-test; *p<0.05, **p< 0.01, ***p<0.001

10 (±SEM).

12 Figure 2. Iron overload leads to high ROS stress in SDHB deficient PCPG cells.

13 A. Cellular ROS levels in SDHBWT and SDHBKD MPC cells were measured by the ROS-Glo

14 H2O2 assay.

15 B. The MitoSOX-Red signal in SDHBWT and SDHBKD MPC cells was measured by flow

16 cytometry, and fluorescence intensity was quantified.

17 C. The ARE transcriptional activity in SDHBWT and SDHBKD MPC cells was measured by

18 a luciferase reporter assay.

19 D. Cellular ROS levels in SDHBWT and SDHBKD MPC cells treated with DFO were

20 measured by the ROS-Glo H2O2 assay. DFO was used as an iron chelator.

21 E. Cellular ROS levels in SDHBWT and SDHBKD MPC cells with siRNA silenced TF was

22 measured by the ROS-Glo H2O2 assay.

1 F. Cellular ROS levels in SDHBWT and SDHBKD MPC cells treated with exogenous Fe2+,

3+ 2 Fe and DFO were measured by the ROS-Glo H2O2 assay.

3 G. Cell viabilities in SDHBWT and SDHBKD MPC cells treated with exogenous Fe2+, Fe3+

4 and DFO were measured by trypan blue exclusion assay. All significance in

5 differences was determined by t-test; *p<0.05, **p< 0.01, ***p<0.001 (±SEM).

7 Figure 3. Pharmacologic ascorbic acid induces ROS overload in SDHB-low cells.

8 A. Cellular ROS levels in SDHBWT and SDHBKD MPC cells under ascorbic acid treatment

9 were measured by the ROS-Glo H2O2 assay. Exogenous ROS scavengers restored ROS

10 levels. Asco, Ascorbic acid; NAC, N-acetyl-L-cysteine; Cata, Catalase. Ascorbic acid,

11 catalase, and NAC concentrations in this and other panels was 1 mM, 500 U/mL, and

12 2.5 mM, respectively. Cells were treated with NAC or catalase for 18 hr.

13 B. The ARE luciferase activity in the presence of ascorbic acid was measured in

14 SDHBWT and SDHBKD MPC cells by a reporter assay. Exogenous ROS scavengers

15 restored ARE activity to the normal level.

16 C. The MitoSOX-Red signal was measured in SDHBWT and SDHBKD MPC cells under

17 ascorbic acid treatment by flow cytometry. Exogenous ROS scavengers restored the

18 MitoSOX-Red fluorescence signal to normal levels.

19 D. ROS levels were measured in SDHBWT and SDHBKD MPC cells under ascorbic acid

20 treatment by MitoSOX-Red staining. Bar = 10 m.

21 E. Cellular ROS levels under exogenous Fe2+, Fe3+, and ascorbic acid treatment were

WT KD 22 measured in SDHB and SDHB MPC cells by the ROS-Glo H2O2 assay. All

1 significance in differences was determined by t-test; *p<0.05, **p< 0.01, ***p<0.001

2 (±SEM).

4 Figure 4. Ascorbic acid leads to more oxidative DNA damage and cellular injury in oxidative-

5 stress-vulnerable SDHB-low PCPG cells.

6 A. Dose-response curves of SDHBWT and SDHBKD MPC cells to ascorbic acid treatment

7 were measured by the CCK8 assay. The cells were treated with ascorbic acid for 24

8 hr. Data was fit for nonlinear regression.

9 B. 8-OH-dG levels in SDHBWT and SDHBKD MPC cells after ascorbic acid treatment were

10 measured by an oxidative DNA damage ELISA assay. 8-OH-dG, 8-

11 hydroxydeoxyguanosine. NAC or catalase was supplemented for 18 hr.

12 C. Mitochondrial DNA PCR assay to measure the amplification of long and short

13 fragments from mitochondrial DNA in SDHBWT and SDHBKD MPC cells after ascorbic

14 acid treatment.

15 D. DNA fragmentation in SDHBWT and SDHBKD MPC cells after ascorbic acid treatment

16 was measured by electrophoresis. ROS scavengers restored the DNA fragmentation

17 induced by ascorbic acid.

18 E. DNA fragmentation (Comet tail) in SDHBWT and SDHBKD MPC cells after ascorbic acid

19 treatment was measured by comet assay. ROS scavengers restored the DNA

20 fragmentation induced by ascorbic acid.

21 F. Quantification of comet assay shown in figure 4E by measuring tail moment.

22 G. Caspase 3/7 activity in SDHBWT and SDHBKD MPC cells after ascorbic acid treatment

23 was measured by a caspase 3/7-Glo assay.

1 H. Apoptosis in SDHBWT and SDHBKD MPC cells after ascorbic acid treatment was

2 measured by Annexin V/PI staining and analyzed by flow cytometry.

3 I. Quantification of the fraction of apoptotic cells in figure 4H.

4 J. Cell viability in SDHBWT and SDHBKD MPC cells after ascorbic acid treatment was

5 measured by the CCK8 assay. ROS scavengers rescued the cell viability of SDHBKD

6 cells.

7 K. Colony formation assay in SDHBWT and SDHBKD MPC cells after ascorbic acid

8 treatment (left panel) and quantification of colony number (right panel).

9 L. Trypan blue exclusion cell counting assay in 100,000 SDHBWT and SDHBKD MPC cells

10 after 3 dose of ascorbic acid treatment. 0.3mM (6 pmol/cell), 1mM (20 pmol/cell),

11 1.8mM (36 pmol/cell). All significance in differences was determined by t-test;

12 *p<0.05, **p< 0.01, ***p<0.001 (±SEM).

14 Figure 5. Ascorbic acid delays SDHB-mutated allograft in vivo.

15 A. Luciferase imaging showing representative SDHBWT (n=10 for each group) and

16 SDHBKD (n=10 for each group) hepatic lesions in vivo under ascorbic acid treatment.

17 The saline-treated group was shown as the control. Asco, ascorbic acid.

18 B. Quantification of tumor volume shown in figure 5A.

19 C. Kaplan–Meier analysis showing overall survival of tumor-bearing animals under

20 ascorbic acid treatment (SDHBWT, p=0.5168; SDHBKD, p=0.0014).

21 D. Levels of oxidative DNA damage in ascorbic acid-treated hepatic lesions from animal

22 models were measured by an 8-OH-dG ELISA assay. Allograft tissues (n=3 for each

1 group) were analyzed. All significance in differences was determined by One-way

2 ANOVA; *p<0.05, **p< 0.01, ***p<0.001 (±SEM).

3 E. Expression of Ki67 and H2A.X in metastatic lesions under ascorbic acid treatment

4 were measured by an immunohistochemistry assay (n=4 for each group). Bar=50

5 m.

6 F. Quantification of immunohistochemistry staining in figure 5E based on integrated

7 optical density. Significance in differences was determined by t-test; *p<0.05, **p<

8 0.01, ***p<0.001 (±SEM).

10 Figure 6. Schematic of the molecular mechanism of the ascorbic acid anti-cancer effect.

11 SDHB-low PCPGs exhibit an imbalance in intracellular iron levels due to abnormal

12 activation of the HIF signaling pathway, which further leads to increased vulnerability to

13 oxidative stress. Targeting of the ROS synthetic pathway with pharmacologic doses of

14 ascorbic acid could be a valuable therapeutic strategy for Cluster I PCPGs with Complex II

15 deficiencies.

1 Abbreviations

2 PCPGs, pheochromocytomas and paragangliomas; SDHB, succinate dehydrogenase B

3 subunit; ROS, reactive oxygen species; TF, transferrin; TFR2, transferrin receptor 2; DMT1,

4 divalent metal transporter 1; VHL, von Hippel-Lindau; HIF1A, hypoxia-inducible factor 1;

5 HIF2A, hypoxia-inducible factor 2; FH, fumarate hydratase; RET, ret proto-oncogene; NF1,

6 neurofibromatosis type 1; MAX, MYC associated factor X; TMEM127, transmembrane

7 protein 127; KIF1B, kinesin family member 1B; ChIP, chromatin immunoprecipitation; ARE,

8 antioxidant response element; DFO, deferoxamine; MPC, Mouse pheochromocytoma;

9 hPheo1, human pheochromocytoma precursor cells; NAC, N-acetyl-L-cysteine; PBS,

10 phosphate-buffered saline; RLU, relative luminescence units; 8-OH-dG, 8-

11 hydroxydeoxyguanosine; FFPE, Formalin-Fixed Paraffin-Embedded.

13 Acknowledgments

14 This research was supported by the Intramural Research Program of the Center for Cancer

15 Research, National Cancer Institute, the Eunice Kennedy Shriver National Institute of Child

16 Health and Human Development, and the Intramural Research Program of the National

17 Institute of Diabetes and Digestive and Kidney Diseases. J.N and K.V were supported by the

18 Czech Science Foundation (18-10832S).

20 Author contributions

21 Y.L. and Y.P. supervised and performed experiments and wrote the manuscript. V.C. helped

22 with the experiments and animal modelling. B.Z. and T.T.H. helped with data analysis. K.P.

23 and C.Y. designed and supervised experiments and contributed towards writing the

1 manuscript, with input from all authors. J.N. and M.L. proposed experiments and

2 contributed to writing the manuscript. D.T., K.H.V., and H.G. contributed towards writing

3 the manuscript.

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Figure 1

A MPC hPheo1 B C WT KD WT KD SDHB MPC SDHBWT β-actin WT KD MPC SDHBKD ** MPC hPheo1 SDHB SDHB HIF-1α ** *** *** * * HIF-2α TF

TFR2

DMT1 Relative protein level SDHB/Actin (Fold change) SDHB/Actin (Fold change) β-actin WT KD WT KD HIF-1α HIF-2α TF TFR2 DMT1 hPheo1 SDHB SDHB SDHB SDHB D

E F G NAM CI CII of TF IHC opitical density DMT1 TF expression TFR2 expression HIF2A expression HIF2A HIF1A expression HIF1A of TFR2 TF IHC opitical density TFR2 of DMT1 EPO expression LDHA expression LDHA TFRC expression SLC11A2 expression SLC11A2 IHC opitical density

H I J K L siCont siTF-1 siTF-2 TF β- ** actin *** *** *** WT SDHB siControl siHIF1 α -1 siHIF1 α -2 siHIF2 α -1 siHIF2 α -2 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer SDHBKD Research. Author Manuscript Published OnlineFirst on March 9, 2020; DOI: 10.1158/1078-0432.CCR-19-2335 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2 A B C D MitoSOX ARE-Luc (%) Fluorescence intensity (%)

E F G SDHBWT SDHBKD ** *** * ***

Fe2+ - + + - - - + + - - Fe3+ - - - + + - - - + + Downloaded from clincancerres.aacrjournals.org on OctoberDFO - - 2, + -2021. + - - © + -2020 + American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 9, 2020; DOI: 10.1158/1078-0432.CCR-19-2335 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3

A B C

D E

SDHBWT SDHBKD PBS Asco PBS Asco MitoSOX DAPI MitoSOX

Figure 4 A B C

SDHBWT SDHBKD Asco - + + + - + + + NAC - - + - - - + - Cata - - - + - - - + mt DNA long fragment mt DNA short fragment

D SDHBWT SDHBKD E SDHBWT SDHBKD F Asco - + + + - + + + ** NAC - - + - - - + - DMSO *** *** Cata - - - + - - - +

Asco Tail moment Tail

NAC Asco - + + + - + + + NAC - - + - - - + - Cata - - - + - - - + SDHBWT SDHBKD Cata

G H I J

Control Asco Asco+NAC Asco+Cata 4.40% 10.6% 4.40% 3.26% WT

SDHB 3.34% 4.67% 1.08% 0.54% 2.58% 38.9% 5.78% 3.23% KD

SDHB 1.70% 9.44% 2.48% 1.11% PI Annexin V

K L Control Asco Asco+NAC 0.3 mM Asco 1 mM Asco 1.8 mM Asco

WT KD WT *** SDHB SDHB *** SDHB MPC KD Colony number (%) Asco - + + - + +

SDHB NAC - - + - - + Downloaded from clincancerres.aacrjournals.orgSDHBWT SDHBKD on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 9, 2020; DOI: 10.1158/1078-0432.CCR-19-2335 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 5 A MPC SDHBWT MPC SDHBKD Saline Asco Saline Asco 23 23 45 456 Time (weeks) Time Time (weeks) Time

B MPC SDHBWT MPC SDHBKD C MPC SDHBWT MPC SDHBKD

15 Saline p = 0.5168 RLU)

7 Asco 10

× 10

Bioluminescence ( Bioluminescence 1 2 3 4 5 Time (Weeks) D E Saline Asco F *** Ki67 γH2A.X ** * Ki67 8-OH-dG (%)

123 123 123 123 Saline Asco Saline Asco γ H2A.X WT KD SDHB DownloadedSDHB from clincancerres.aacrjournals.org on October 2, 2021. ©Saline 2020Asco American AssociationSaline Asco for Cancer Research. Author Manuscript Published OnlineFirst on March 9, 2020; DOI: 10.1158/1078-0432.CCR-19-2335 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 6

SDHBWT SDHBKD (Cluster II) (Cluster I)

SDHB SDHB Succinate Fumarate Succinate

ROS Fe HIF-1α ROS Pharmacologic Ascorbate Fe EGLN1 Fe ROS EGLN1 TF TF Fe Fe HIF-1α Fe Fe TF Fe HIF-1α Pseudohypoxia TF Ub Fe TF Ub Iron Ub TF Ub Overload TFR2 TFR2 Fe HIF-1α DMT1 Fe

Proteasomal Degradation

Therapeutic targeting of SDHB-mutated pheochromocytoma/paraganglioma with pharmacologic ascorbic acid

Yang Liu, Ying Pang, Boqun Zhu, et al.

Clin Cancer Res Published OnlineFirst March 9, 2020.

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