Tumor Treating Fields (TTFields) Delay DNA Damage Repair Following Radiation Treatment of Glioma Cells Implications for Irradiation through TTFields Transducer Arrays M. Giladi1, M. Munster1, R. S. Schneiderman1, T. Voloshin1, Y. Porat1, Z. Bomzon1, E. Kirson1, U. Weinberg1, Y. Palti1, and M. D. Story2

1Novocure, Haifa, Israel, 2University of Texas, Southwestern Medical Center, Dallas, TX

Research funding: Novocure Galera Therapeutics

Disclosures

Tumor Treating Fields

• Tumor Treating Fields (TTFields) are an effective anti-neoplastic treatment modality

• Delivered via application of low intensity, intermediate frequency, alternating electric fields using insulated ceramic transducer arrays placed directly on the scalp in the region surrounding a tumor

Patient receiving TTFields therapy for GBM

EF-14 Phase 3 Pivotal Trial - A Prospective, Multicenter Trial of TTFields Together With Temozolomide Compared to Standard-of-Care Temozolomide Alone in Patients With Newly Diagnosed GBM

TTFields Anti-Mitotic Mechanism of Action NSCLC

Ovarian

Ovarian

HeLa Tubulin-GFP

Adapted from Giladi et al Sci Rep, 2015; Dec 11;518046 Adapted from Gera et al. PLoS One. 2015; May 26;10(5):e0125269 Voloshin T., et al. Int J Cancer. 2016 Aug 25. doi: 10.1002/ijc.30406. Adapted from Schneiderman AACR 2014 Adapted from Porat SNO 2014

HeLa Tubulin-GFP

TTFields Enhance Radiation and Bleomycin Treatment Efficacy by Delaying DNA Damage Repair

U118 (Glioma)

Dose (Gy)

TTFields Treatment After Radiation Delay γH2AX Foci Resolution

Control TTFields IR + TTFields IR

1h

2h

24h

*<0.01, **<0.001. RT, Radiation treatment.

TTFields Induce DNA Damage and Slow the Repair of Radiation-Induced DNA DSBs in NSCLC Cell Lines

TTFields Cause the Downregulation of the BRCA/FANC Family of DNA Repair Genes

• R-loops can cause: • Genomic instability • Chromosomal rearrangements • DNA SSBs and DSBs

• FANC genes protect DNA within replication forks • Downregulation can result in:

• replication fork stalling and collapse • R loops

• Gene expression analysis identified: • downregulation of BRCA/FANC and ATM pathways • increased DNA damage cell cycle checkpoint controls

Responsive cells Less responsive cells

Extended Exposure to TTFields Causes Shortened DNA Replication Strands

C o n tro l-H 1 5 7

T T F -H 1 5 7

DNA fiber length (µm)

% F

requ

ency

DNA fiber length (µm)

% F

requ

ency

60h

2 4 6 8 1 0 1 2 1 4 1 6 1 80

1 0

2 0

3 0

4 0

5 0

15h

2 4 6 8 1 0 1 2 1 4 1 60

1 0

2 0

3 0

4 0

5 0

30h

2 4 6 8 1 0 1 2 1 4 1 60

1 0

2 0

3 0

4 0

5 0

45h

2 4 6 8 1 0 1 2 1 4 1 60

1 0

2 0

3 0

4 0

C o n tro l-H 1 2 9 9

T T F -H 1 2 99

0 2 4 6 8 1 00

1 0

2 0

3 0

4 0

5 0

15h

0 2 4 6 8 1 00

2 0

4 0

6 0

8 0

30h

0 2 4 6 8 1 0 1 2 1 40

1 0

2 0

3 0

4 0

5 0

60h

0 2 4 6 8 1 0 1 20

1 0

2 0

3 0

4 0

5 0

45h

Extended Exposure to TTFields Results in the Production of R-loops

C o n tr o l T T F ie ld s0

2 0

4 0

6 0

8 0

1 0 0

Me

an

in

tes

nit

y p

er

nu

cle

us

*

C o n tr o l T T F ie ld s0

2 0

4 0

6 0

8 0

1 0 0

Mea

n in

tesn

ity

per

nu

cleu

s

*

R-loops IF- H157- 72h R-loops IF- H1299- 72h

TTFields Enhance the Therapeutic Efficacy of Radiation or Other Agents that Cause or Interfere with DNA Replication or Repair

• Short TTFields Exposure – Reduction in DNA repair gene

• transcription • translation

– Production of DNA lesions – Inhibition of DNA repair

• Radiation • Cisplatin • Other agents

• Long TTFields Exposure – Reduction in DNA repair gene

• transcription • translation

– Replication stress increase • Shortened replicated DNA strands • Stalled and collapsed replication forks

– Production of DNA lesions • R loops • DNA single and double strand breaks

Susceptibility to DNA damaging agents

DIVIDING CELLS TTFields

DNA Damage Repair

DNA Damage

Ionizing Radiation UV Light Exposure Chemical Exposure Cellular Metabolism

TTFields

Anti Mitotic Effects

Failed DNA Replication and Repair

Cell Death

Surviving Cell

Down-regulation of BRCA/FANC genes Replication fork stalling DNA replication errors Collapse of DNA replication forks

TTFields

Failed DNA Repair

Abnormal Cell

Cell Death

Abnormal Cell

Development of R loops Development of SSBs and DSBs

Schematic Diagram of TTFields Mechanisms of Action

Ionizing Radiation Chemical Exposure

Summary

• There is a mechanistic rationale for the development of alternative strategies for the use of TTFields

• The response of skin directly below TTFields ceramic transducers appears to be minimal and unlikely to interfere with combined therapy strategies