Development of the first clinical targeting the DNA repair/redox signaling APE1/Ref-1; Clinical indications for solid tumors and CIPN prevention

Mark Kelley, Ph.D., with Jordan Dew Mark R. Kelley, Ph.D.

• Betty and Earl Herr Professor in Pediatric Research • Professor, Departments of , Biochemistry & Molecular Biology and Pharmacology & Toxicology • Associate Director, Herman B Wells Center for Pediatric Research • Associate Director of Basic Science Research, Mark Kelley, Ph.D. with Owen Indiana University Simon Center • Director, Program in Pediatric Molecular Oncology & Experimental Therapeutics Disclosures:

Chief Scientific Officer and Founder, Apexian Pharmaceuticals

Licensing agreements for and reagents to:

Novus Biologicals Abcam Millipore

Supported by:

The National Institutes of Health, National Cancer Institute CA167291, R21NS091667, Hyundai Hope on Wheels Foundation Grant, Betty and Earl Herr Chair in Pediatric Oncology Research, Hamer Foundation, Jeff Gordon Children’s Research Foundation and the Riley Children’s Foundation. The Target APE1/Ref-1 Overview

• APE1 (apurinic/apyrimidinic endonuclease), also called Ref-1 (redox effector factor 1), is a multifunctional cellular with at least two distinct and separate functions:

• APE1 Redox Function: Redox regulation of transcription factors (TFs) effecting critical aspects of cancer cell survival and growth including HIF-1, STAT3, NF-KB, and — We can target multiple signaling others. pathways relevant to various with one protein— as APE1 regulates • DNA Repair Function: DNA base repair transcription factors (TFs) HIF1a, caused by oxidative stress, alkylating STAT3, NFkB and others. agents, and ionizing radiation

• RNA Degradation and quality control: — APX3330 inhibits only the APE1 Interaction with NPM1 redox signaling activity. • Various cancers, including treatment resistant tumors, have shown elevated expression of APE1 suggesting adaptation and unique survival mechanisms through this pathway. APE1/Ref-1 functions: DNA repair and Redox signaling regulation of TFs

DNA C99 C65 DNA C93 Redox Role damage Repair

T G A A T C AP-1 A C T T A G Glycosylase (Ogg1, Nth1) NFkB (reduced) (oxidized) T G A A T C HIF-1a A C T T A G Ref-1 CREB T G * A T C (oxidized) (reduced) PAX A C T T A G STAT3 Repair APE1 Active Redox control of T G A T C Transcription factors C65 site A C T T A G

5’-P Target expression: T G A A T C Growth A C T T A G DNA Ligase Angiogenesis T G A A T C A C T T A G Alteration of APE1/Ref-1 protein expression has been shown to be elevated in:

1. Non-small cell lung cancer 2. Colorectal cancer 3. Breast cancer 4. Prostate cancer 5. Gynecologic cancers (ovarian, cervical) 6. Pancreatic cancer 7. Glioblastoma multiforme, meduloblastoma 8. Renal cancer 9. Gastric cancer 10. Germ cell tumors 11. Head-and-neck cancers 12. Multiple myeloma (hematologic cancer) 13. Osteosarcoma and Rhabdomyosarcoma (pediatric) The Drug

E3330 = APX3330

• APX3330 was originally developed by Eisai (E3330) as a NFkB-TNFa inhibitor for the treatment of inflammatory .

• Eisai ended APX3330 development after in-licensing Revovir® (clevudine) for the treatment of hepatitis B and Humira (adalimumab) for treatment of rheumatoid arthritis, IBD and other indications. APX3330 Continued

• The drug has a direct and selective interaction with APE1 as demonstrated by chemical footprinting, mass spectrometry, and other biochemical data.

• Although multiple pathways may be modulated, unacceptable toxicity following APE1 inhibition has not been observed in animal or human studies.

• Preclinical data supports the use of the drug as a single agent; future directions indicate partnering APX3330 with various clinical agents such as JAK2 inhibitors (Ruxolitinib, LY3009104. etc), STAT3 inhibitors, gemcitabine and Abraxane (nab-paclitaxel). APX3330 inhibits APE1 Redox Function Blocking TF Activity

APX3330

(reduced APE1) (oxidized TF) C65 HIF-1a NFkB TF C93 STAT3 X AP-1 Redox NRF2 X active site (oxidized APE1) (reduced TF)

(oxidized APE1) (reduced TF binds to target DNA)

X Target : Growth TF Inflammation AngiogenesisX APE1 cysteines involved Disulfide in redox function bonds C99 65-93 65-99 93-99

C93

C65

Courtesy of Dr. Millie Georgiadis, collaborator

• Kelley MR, Luo M, Reed A, Su D, Delaplane S, Borch R F, Nyland II RL, Gross ML, Georgiadis M. (2011) Functional analysis of new and novel analogs of E3330 that block the redox signaling activity of the multifunctional AP endonuclease/redox signaling enzyme APE1/Ref-1. Antioxid Redox Signal. April; 14(8): 1387-1401. • Su D, Delaplane S, Luo M, Rempel D, Vu B, Kelley MR, Gross ML, Georgiadis M. (2011) Interactions of APE1with a redox inhibitor: Evidence for an alternate conformation of the enzyme. Biochemistry. 50(1): 82-92. • Luo M, Zhang J, He H, Su D, Chen Q, Gross M, Kelley MR, Georgiadis, M. (2012) Characterization of the Redox Activity and Disulfide Bond Formation in Apurinic / Apyrimidinic Endonuclease. Biochemistry. Jan 17; 51(2):695-705. • Zhang J, Luo M, Marascot D, Logsdon D, LaFavers KA, Chen Q, Reed A, Kelley MR, Gross ML, Georgiadis MM. (2013) Inhibition of Apurinic/apyrimidinic endonuclease I’s redox activity revisited. Biochemistry. Apr 30;52(17):2955-66 Evolution of the C65 redox center in APE1

Zebrafish Stickleback

Lizard X. tropicalis Platypus Mouse Opossum Dog Chicken Ape1 is also missing Horse the redox C Rhesus Human

Human CCCCCCC Horse CCCCCCC X. tropicalis SCCKCCC Rhesus CCCCCCC Opossum CCCCCCC Stickleback TCCECCC Mouse CCCCCCC Platypus CCCRCCC Zebrafish TCCECCC Dog CCCCCCC Lizard TCCKCCC APX3330 blocks the activity of primary Cys residues required for APE1 redox function wtAPE1 C65/C93/C99 C65/C93 C65/C99

APX3330 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 uM

120

wt-Ape1 100 C65/C93/C99 C65/C93 Labeling indicates only the Cys * C65/C99 80 present

60 *

40 *

AP1 DNA-binding intensity (%) intensity AP1 DNA-binding 20

0 -10 0 10 20 30 40 50 E3330 (uM) Mass spectra of APE1 after incubation without (A) and with APX3330 (B) in the presence of NEM for 30 min (left panel) and 6 h (right panel). (A) Δ40APE1+2NEM Δ40APE1+2NEM 32252 32252 Δ40APE1/NEM 100 100 Δ40APE1/NEM 30 min 6 h

% % ApeΔ40+3NEM 32377 * * 0 mass 0 mass 32000 32200 32400 32600 32800 32000 32200 32400 32600 32800 (B) Δ40APE1+2NEM Δ40APE1+2NEM Δ40APE1/3330/NEM 32252 32254 100 Δ40APE1/3330/NEM 100 6 h 30 min

% % Δ40APE1+7NEM 32879 Δ40APE1+3NEM 32379 * 0 mass 32000 32200 32400 32600 32800 0 mass 32000 32200 32400 32600 32800

No 3330, NEM specifically modifies APE1 resulting in the formation of a +2 NEM = C99 C138 (solvent accessible). +3330 = 7NEM appearance = all cys now accessible

Su D, Delaplane S, Luo M, Rempel D, Vu B, Kelley MR, Gross ML, Georgiadis M. (2011) Interactions of APE1with a redox inhibitor: Evidence for an alternate conformation of the enzyme. Biochemistry. In Press Using HDX mass spectrometry, APX3330 interacts with and inhibits the redox activity of APE1 at two regions.

These results suggest that APX3330 destabilizes APE1’s structure rather than stabilizing it.

Interaction of APX3330 with APE1 as detected by HDX mass spectrometry. (A) HDX data are shown for peptides with slower exchange rates in the presence of 1.6 mM E3330 (□) as compared to the exchange rates in the absence of compound (■). (B) The peptides that showed protection from deuterium exchange are shown highlighted on the structure of APE1. Residues 68– 74 are colored orange and residues 266–273 magenta. Shown as stick models are R73 (orange) and R177 (blue), two Arg residues in the proximity of the regions of interaction identified by HDX mass spectrometry.

Published in: Jun Zhang; Meihua Luo; Daniela Marasco; Derek Logsdon; Kaice A. LaFavers; Qiujia Chen; April Reed; Mark R. Kelley; Michael L. Gross; Millie M. Georgiadis; Biochemistry 2013, 52, 2955-2966. Supporting Drug Selectivity Data

180

160

140 Vector cont 120 + wtwt Ref-Ref-1-1 100

80

% Survival % 60

40

20

0 Cont APX3330 Inhibition of APE1/Ref-1 with APX3330 Blocks TF Function and Downstream Factors

NFkB AP-1 HIF1a STAT3 (fos/JUN)

DNA

Luciferase Stat3

APX3330 APX3330 uM APX3330 uM APX3330 uM High Unmet Clinical Need for Pancreatic Cancer

• In 2015, 48,960 Americans will be diagnosed with pancreatic cancer and more than 40,560 will die from the disease. Pancreatic cancer 1-year survival rates are ~25% and 5-year survival rates are ~7%. • Pancreatic cancer thrives in an inflammatory, hypoxic, and dense/stromal microenvironment making it hard to treat. Few patients are diagnosed at an early stage leading to an average life expectancy following diagnosis of 3 to 6 months. • Currently approved chemotherapeutic treatments include combination and single-agent use of paclitaxel, cisplatin, gemcitabine (Gemzar®), and 5-flurouracil. Other approved treatments (Abraxane, FOLFIRINOX) have high toxicities limiting use to patients that can tolerate the . • Pancreatic cancer represents an area of high unmet clinical need with Breakthrough regulatory approval potential for even modest improvements in survival.

SOURCE: American Cancer Society website APE1 Expression is Linked to Poor Survival in Pancreatic Patients

Early stage patients undergoing Whipple procedure TCGA/PDAC Patients Median Survival Cohort (p<0.0003) High Ref-1/APE 1 N = 9 4 months (Red)

Low Ref-1/APE1 N = 61 16.8 months (Blue) Pancreatic cancer patient tumors express APE1/Ref-1 in both tumor and stroma Patient # 1 2 3

A

18 16 Tumor * 14 Stroma C 12 n=10 10 * 8 B 6 4 2 Positive pixels / 10^4 0 Weak Positive Strong Jiang Y, Zhou S, Sandusky GE, Kelley MR, and Fishel ML. (2010). Cancer Investigation 28 (9): 885-895 positive positive Stroma Signaling E3330 E3330 Tumor E3330 Ref-1

Ref-1 expression in panc patient sample Pro-Angiogenic Factors HIF-1a AP-1 NFkB VEGF IL8 E3330

Pancreatic Angiogenesis / cancer cells Invasion / Metastases Tumor-assoc. NFkB macrophages Tumor Microenvironment Stellate Cells (Angiogenesis/Invasion/ Cancer Associated Metastases) Fibroblasts (CAFs) Tumor + CAFs (1:4)

(Pa03C & 1301-63 hTERT-GFP) Addition of CAFs to tumors accelerates tumor growth rate in vivo

2000.0 1800.0 Tumor only 1600.0 3 1400.0 1:2 1200.0 1000.0 1:4 800.0 600.0

Tumor volume mm400.0 1.80 200.0 1.60 * * 0.0 1.40 8 10 14 17 21 24 1.20 Day after inoculation 1.00 0.80

0.60

Tumor weight at sac (g) 0.40

0.20

0.00 Tumor only 1:2 1:4 0 20 30 40 µM APX3330 10.05 C Pa03

PDAC (Tumor) Cells Pa03C

APX3330 DMSO 20 30 40 µM APX3330 alone Pa03C - cultures CAF co CAF Pa03C+

PDAC Cells + CAFs Gem + APX3330 in 3D model

Tumor + CAFs Tumor only Tumor CAFs Cntl 1.25 2.5 5 Cntl 1.25 2.5 5 Cntl 1.25 2.5 5 Gem nM

Gem +APX

* * * * * * * * APX3330 Reduces Tumor Growth and Metastasis

PaCa-2

Human PDAC Number of lymph node metastases Metastasis

18

16 Individual Mice 14 Average

12

10

8 6 * 4

2

# of Lymph Node Metastases Node Lymph of # * p<0.01 0 Vehicle E3330APX3330

1SOURCE: Fishel ML, Jiang Y, Rajeshkumar NV, Scandura G, Sinn AL, He Y, Shen C, Jones DR, Pollok KE, Ivan M, Maitra A, Kelley MR. (2011). Impact of APE1/Ref-1 Redox Inhibition on Pancreatic Tumor Growth. Molecular Cancer Therapeutics. Sep;10(9):1698-708. Endothelial cell tumor growth is Ape/ref-1 dependent

Redox function of Apex-1 is required for MCP-1 activation and EC tumor growth in vivo. Apex-1 knockdown in EOMA cells resulted in significant decrease in MCP-1 reporter activity (A), and MCP-1 release in the media was measured by ELISA (B). C: MCP-1 reporter activity was significantly decreased in c-Jun knockdown EOMA cells and in E3330 (50 µM, 5 h)-treated cells (D). Redox changes of Apex-1 influences HE outcome in vivo. E: tumor growth rates were evaluated after 7 days of E3330 treatment (25 mg/kg ip twice daily) alone and in combination with CRT0044876 (10 mg/kg ip twice daily). F: tumor volume was quantified using calipers (length × width × height). Results are expressed as means ± SD; *P < 0.05.

Ayan Biswas et al. Am J Physiol Cell Physiol 2015;309:C296-C307 Clinical Plans for APX3330

• Apexian will complete a two-part phase I oncology study: • Increasing doses in patients with treatment-refractory solid tumors • 20-40 patients

• Study endpoint: • Identify the RP2 dose of APX3330 • Based upon • of the agent • evidence of anti-tumor effect • pharmacokinetic and pharmacodynamic

• IND accepted by the FDA: • All study documents are ready and sites identified • Contracts pending completion of the funding round

• Additional safety, tolerability and efficacy POC Academic – Industry / Biotech Partnership

Bench to Bedside

Bench Findings: A Phase I Clinical Trial Inhibition of Ref-1 via Open-Label Dose APX3330 reduces tumor Escalation Study of Oral burden in mice APX3330 in Subjects (reduced) (oxidized) HIF-1a Target APE1/Ref-1 Ref-1 with Advanced Solid (oxidized) STAT3 X (reduced) Tumors

Redox control of Transcription factors

Target gene: Growth, Inflammation, Angiogenesis Inhibitor: APX3330 IND approved July 16, 2016 IU IRB approved Aug 22, 2016 B C A Schematic of APX3330. Groups to be further investigated include the

(A) Quinone series, D (B) 3-Position series, (C) Alkyl Sidechain series and (D) Carboxylic Acid/Amide series.

APX3330 APX2009 CURRENT PIPELINE APX2014 APX2050 APX2048 APX 3330APX 3330APX APX2009 combinations APX3330+ APX 3330APX APX 3330APX Pipeline and Indications Chemo Discovery Discovery Discovery Discovery Age- Ape1/Ref Related Macular Degeneration - Induced Peripheral Neuropathy Cancer Liquid Tumors - 1 Diseases - Multiple Preclinical Development Solid Tumors – ALL – Pancreatic Cancer Filing IND Phase 1 Continuing to Follow the Science

Prostate Ovarian

Pancreatic Cancer Leukemia

Combinations “Repair” “Redox” APE1 APX3330 Function Function

Other New Diseases Molecules Example pathways that are altered in low passage patient derived PDAC cells following APE1 knockdown and Fluidigm C1 single cell sorting-RNA seq analysis. Validation of Single Cell KD & Potential Use for Drug Development/Specificity

N S R

N S R N S R APE1 Complexes with HIF1α & STAT3 under Hypoxia

Endogenous APE1

10.05 Pa03C A IP: Input IgG APE1 B IP: Input IgG APE1 N: Normoxia N: Normoxia N H N H N H N H N H N H H: Hypoxia H: Hypoxia WB: HIF1a WB: HIF1a

STAT3 STAT3

APE1 APE1

NFkB NFkB

APE1 APE1 IL-6 targeted APX3330 agents; Stromal%Cells% Siltuximab, IL#6% CA9 Toxilizumab

JAKX STAT3 pH X JAK2 inhibitors; Stabilization X Ruxolitinib, HIF1a X Pacritinib, etc STAT3 Inhibitors X X X Bcl2, Survivin, VEGF CA9 VEGF X STAT3 TGF-B, Bcl-xL, HIF1a HIF, etc.

DNA

Bevacizumab NFKB

X TNFa IL6, etc Stromal%Cells% IL#6%

JAK

STAT3 X APX3330 X STAT3 Inhibitors PG-S3-001 DR-4-89 STAT3

DNA STAT3 DNA binding is redox sensitive and can be stimulated by APE1/Ref-1….and inhibited by APX3330 APX3330

STAT3 APE1/ Tumor Ref-1 APE1/ mass Ref-1 - + APX3330

p-STAT3

T-STAT3

+ IL-6

* Basal ** ** ** STAT3 DNA binding (%)

E3330 (µM) / Signaling Hypoxia Stromal%Cells% Inflammation IL#6%

JAKX STAT3 X Ruxolitinib APX3330 X

STAT3

DNA Pa03C cells in 3D: Combo Ref-1 inhibition + Jak2 inhibition

Tumor area – Co-culture 2500000 Tumor area – Tumor alone 2000000 Rux only

1500000 Rux+APX uM^2

1000000 CAF area – Co-culture 500000

0

Ruxolitinib (uM) Cytokine / Growth factor Signaling Hypoxia Inflammation

CAIX

X SLC-0111 HIF1a APX3330 X CAIX X HIF1a

DNA Hypoxia-Induced CA9 mRNA: Inhibiton by Ref-1 KD and APX3330

1.2

1.0

0.8 Ctrl

0.6

Hypoxic Hypoxic 0.4 Fold change from

0.2

0.0 SCR siRef-1 DMSO APX3330 Pa03C APX3330 PDAC Cells + CAFs DMSO 20 30 40 µM

APX3330

APX3330

Pa03Calone + SLC-0111 -

APX3330

cultures APX3330 + SLC-0111 Pa03C + CAF co Pa03CCAF + Pa03C cells in 3D: Combo Ref-1 inhibition + CA9/12 inhibition

Area Intensity

Tumor area – Co-culture Tumor area – 2000000 Tumor area – Co-culture 1800000 Tumor alone 70000000 Tumor area – Tumor alone 1600000 E3330 60000000 1400000 50000000

2 E3330 +SLC-50 1200000 40000000 +SLC-50 1000000 uM 800000 30000000 CAF area – RFU CAF area – 600000 20000000 Co-culture Co-culture 400000 10000000 200000 0 0

APX3330 (uM) APX3330 (uM) 3330

HIF1a X

X VEGF X HIF1a

DNA

Bevacizumab Cancer that produce CIPN Sensory neuropathy with symptoms such as:

ü distal parasthesias (tingling, numbness, burning sensations) ü altered proprioception (awareness of position of one’s body) ü coldness in extremities ü acute/chronic pain

Chemotherapy effects motor neurons less frequently than sensory neurons

Autonomic nervous system dysfunction (palpitations, orthostatic hypotension, impotence) is rarely seen

M. Vasko et al. “The Role of DNA Damage and Repair in Neurotoxicity caused by Cancer Therapies” in DNA Repair in Cancer Therapy: Molecular Targets and Clinical Applications. (2012) Kelley, M., Ed. Academic Press/Elsevier Induced Peripheral Neuropathy (CIPN) Associated with CIPN

• Platinum compounds (cisplatin, carboplatin, oxaliplatin) • Vincristine • Taxanes (docetaxel, paclitaxel) • Epothilones (ixabepilone) • Bortezomib (CIPN occurs in 37%–44% of patients with multiple myeloma) • Thalidomide (CIPN develops in 20%–40% of patients) • Lenalidomide

Overall, 40% of patients receiving cisplatin and taxol develop CIPN!

A cross-sectional study of patients with testicular cancer re-evaluated 23–33 years after finishing treatment showed that CIPN remains detectable in up to 20% of patients, being symptomatic in 10% of them.

The combination of 5-FU and oxaliplatin is frequently used in patients with gastrointestinal cancer, and 92% of patients develop sensory CIPN.

Moya del Pino, Brittany. A Closer Look: Chemotherapy-induced Peripheral Neuropathy. NCI Cancer Bulletin, February 23, 2010. http://www.cancer.gov/aboutnci/ncicancerbulletin/archive/2010/022310/page6 Patients used analogies to describe symptoms (Tanay et al. 2016)

• ‘Severe buring in fingertips’, ‘Like putting them (fingers) on hot stove’, • ‘A strip of numbness across fingers’ (Boehmke & Dickerson 2005)

• ‘Like fingernails on a chalkboard’, ‘Pain like needle stuck in my toes’ • (Bakitas 2007)

• ‘Walking on hot coals’, ‘Walking on a rock on the bottom of your feet’, ‘Sandpaper at the bottom of your feet’, ‘Something crawling’ • (Tofthagen 2010b)

• ‘Walking in mud’, ‘Bunched up socks’, ‘Walking on sandpaper’, • ‘Getting a cast off’, ‘Blob of numbness’, ‘Feet are asleep’ • (Speck et al. 2012) Estimates of the number of adult patients Uses of platinum agents in treated annually with either cisplatin, pediatric oncology: oxaliplatin or carboplatin are approximately > 200,000 a year: Cisplatin and Carboplatin are ü 50,000 patients with metastatic used in: colorectal cancer

Neuroblastoma ü 20,000 with stage III colon cancer Germ cell tumors Osteosarcoma ü 12,000 with pancreatic cancer Hepatoblastoma ü 25,000 with gastroesophogeal, and

Brain tumors ü 10,000 with head and neck cancer. Retinoblastoma ü 4,000 with ovarian cancer

ü etc Oxaliplatin is not used. Putative sites of neuronal dysfunction following specific anticancer drug treatments, indicated in italics.

Nuclear DNA damage Cisplatin, oxaliplatin, Local DRG carboplatin, inflammation ionizing radiation Taxanes, ROS/RNS oxaliplatin production Taxanes, cisplatin, Disruption of oxaliplatin microtubule dynamics/ axonal transport Taxanes, epothilones, vinca alkaloids, cisplatin, bortezomib

Mitochondrial DNA damage Cisplatin, oxaliplatin IENF degeneration Mitochondrial permeability Taxanes, epothilones, changes vinca alkaloids, Taxanes, cisplatin, bortezomib cisplatin, oxaliplatin, bortezomib DNA Repair in Neurons

Nucleus Mitochondria Direct Repair ????? DNA damage in neurons is repaired using these BER BER pathways: NER NER

MMR MMR

HR / NHEJ ?????

Fishel, ML., Vasko, MR. and Kelley, MR. (2006) DNA repair in neurons: So if they don't divide what's to repair? Invited and peer-reviewed review. Research 614(1-2); 24-36. Oxidative DNA damage and crosslinks induced by the platinum drugs APE1 knockdown effect on cisplatin, oxaliplatin and carboplatin-induced iCGRP release in DRG cells

Basal CAP 30 nM 175 N=12 P<0.05 Basal 150 150 150 125 * * 100 100 100 iCGRP 75 iCGRP iCGRP 50 50

(fmol/well/10min) 50 (fmol/well/10min) (fmol/well/10min) 25

0 0 0 SCsiRNA + + - SCsiRNA + + - SCsiRNA + + -

APEsiRNA - - + APEsiRNA - - + APEsiRNA - - +

Cisplatin 10 µM µ Oxaliplatin 30 M Carboplatin 300 µM Effect of altered Ape1 levels on cisplatin-induced iCGRP release from sensory neuronal cells.

Yanlin Jiang et al. Cancer Res 2008;68:6425-6434

©2008 by American Association for Cancer Research Effect of knocking Ape1 down on cisplatin, oxaliplatin and carboplatin-induced DNA damage (p-H2AX) in DRG cells

A. Cisplatin(50uM) B. Oxaliplatin(300uM) C. Carboplatin(500uM)

Scr siAPE1 Scr siAPE1 Scr siAPE1

0 8 24 48 0 8 24 48h 0 8 24 48 0 8 24 48h 0 8 24 48 0 8 24 48h P-H2AX Actin

10 Scr 6 2.5 Scr Scr 8 siAPE1 5 * * siAPE1 2 siAPE1 Actin Actin 4 Actin to * to 6 to 1.5 3 4 1 2 * ormalized 2 0.5 N 1 Normolized Normolized 0 0 0 0 8 24 48hrs 0hr 8hrs 24hrs 48hrs 0hr 8hrs 24hrs 48hrs N=3 Cisplatin N=4 Oxaliplatin N=3 Carboplatin *p<0.05 *p<0.05 *p<0.05 8oxoG levels in DRG Neuronal Cultures following APE1 KD and cisplatin, oxaliplatin or carboplatin treatments A

B Assessment of Cisplatin-Induced DNA damage

Pt-GpG Pt-GpG Pt-GpG (1, 2-intrastrand (1,3-intrastrand (interstrand crosslink) crosslink) crosslink) >90% <5% <5%

DNA cisplatin conc. Fragmentation 30 uM 10 uM mAb specific to 1,2-Pt-GG DNA slot blot

1,2-Pt-GG G-Pt-G 1,3-Pt-GG Targeted inhibition of APE1 expression in rat neuronal cells significantly reduces removal of Pt-damage

N.T SC-siRNA APE1-siRNA 1 2 3 No CDDP

0 hr Hours after 15 4 hrs uM CDDP treatment for 3 hrs 8 hrs

1,2-Pt damage SYBR Gold (DNA control)

100%

80% damage

- 60%

40% Non Transfection SC siRNA 20% APEI siRNA

Relative Pt Relative 0% 0 2 4 6 8 10 Hours after CDDP treatment Add-back of wt-APE1 restores repair of 1,2-Pt-GpG damage in DRG cells treated with Ape1-siRNA

120% 100%

80% SC siRNA damage - 60% APEI siRNA Pt 40% APEI siRNA+Ad5 vector APEI siRNA+WT-APEI 20%

Relative 0% 0 4 8 12 16 Time (h)

SC- APE1-siRNA______siRNA mock +vector +wt-APEI 1 2 3 4 No CDDP

0 hr Time after 15 uM 4 hrs CDDP treatment for 3 hrs 8 hrs

1,2-Pt damage SYBR Gold (DNA control) Kelley MR Kelley PMCID: PMC4154694 PMCID: oxaliplatin,orcarboplatin induced sensory neuropathy. cultures treated with vehicle and those treated with E3330 using Student's established AP Each column is the mean , Jiang , Y, APX3330 enhances enhances APX3330 DNA APE1 endonuclease repair Guo endonuclease C, Reed C, A, ±

SEM ofSEM the percent increase in endonucleaseAPE1 activity using the % increase in APE1 repair activity Meng assay. 100 105 120 110 115 95 H, VaskoH, (2014)MR. Role theDNAof base excision repair protein, APE1in cisplatin, N= 4 activity in DRG cells activity in An 0 asterisk indicates a statistically significant difference between PlosOne 12.5 APX3330 . Sept4;9(9):e106485.10.1371/journal.pone.0106485. DOI: 25 * ( μ M) 50 * t - test. Peripheral blood flow is regulated by CGRP; i.e. measuring iCGRP release in vitro or blood flow in vivo is indicator of DRG function

Heated Platform

Laser Doppler Probe Digital Thermometer Dermatome L4

Needle (~1mm rostral to probe)

Chemoprotective experiment using APX3330 and cisplatin Cisplatin 3mg/kg APX3330 treatment (25 mg/kg) can reverse cisplatin neurotoxicity in R1 and R2 APX3330 attenuates neurotoxicity induced by systemic administration of cisplatin to neuroblastoma tumor- bearing mice Acknowledgements: • Collaborators ü Michelle Grimard – Research Analyst ü Randy Wireman – Research Analyst • Dr. Melissa Fishel ü Fenil Shah, PhD P -- Postdoctorate • Dr. Millie Georgiadis ü Derek Logsdon – Graduate Student (biochemistry-structure) • Dr. Jill Fehrenbacher (CIPN) • Dr. John Turchi (repair-CIPN) Supported by: • Dr. Michael Vasko (CIPN) • Dr. Travis Jerde (prostate) The National Institutes of Health, National Cancer Institute • Dr. Karen Pollok (in vivo CA167291, NS091667 therapeutics core) Hyundai Hope on Wheels Foundation Grant, Betty and Earl Herr Chair in Pediatric Oncology Research, • Tony Sinn Hamer Foundation, • Dr. Nadia Atallah (PUCCR) Jeff Gordon Children’s Research Foundation and the Riley Children’s Foundation. • Emery T. Goossens (PU-Stats) • Dr. David Jones (CPAC)