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Tumor treating fields cause replication stress and interfere with DNA replication fork maintenance: Implications for cancer therapy

Open AccessPublished:October 21, 2019DOI:https://doi.org/10.1016/j.trsl.2019.10.003
      Tumor treating fields (TTFields) is a noninvasive physical modality of cancer therapy that applies low-intensity, intermediate frequency, and alternating electric fields to a tumor. Interference with mitosis was the first mechanism describing the effects of TTFields on cancer cells; however, TTFields was shown to not only reduce the rejoining of radiation-induced DNA double-strand breaks (DSBs), but to also induce DNA DSBs. The mechanism(s) by which TTFields generates DNA DSBs is related to the generation of replication stress including reduced expression of the DNA replication complex genes MCM6 and MCM10 and the Fanconi's Anemia pathway genes. When markers of DNA replication stress as a result of TTFields exposure were examined, newly replicated DNA length was reduced with TTFields exposure time and there was increased R-loop formation. Furthermore, as cells were exposed to TTFields a conditional vulnerability environment developed which rendered cells more susceptible to DNA damaging agents or agents that interfere with DNA repair or replication fork maintenance. The effect of TTFields exposure with concomitant exposure to cisplatin or PARP inhibition, the combination of TTFields plus concomitant PARP inhibition followed by radiation, or radiation alone at the end of a TTFields exposure were all synergistic. Finally, gene expression analysis of 47 key mitosis regulator genes suggested that TTFields-induced mitotic aberrations and DNA damage/replication stress events, although intimately linked to one another, are likely initiated independently of one another. This suggests that enhanced replication stress and reduced DNA repair capacity are also major mechanisms of TTFields effects, effects for which there are therapeutic implications.

      Abbreviations:

      CI (combination index), DDR (DNA damage response), DSBs (double strand breaks), FA (Fanconi Anemia), GBM (glioblastoma), HSA (highest single agent), IR (ionizing radiation), NSCLC (non–small-cell lung cancer), RPA (replication protein A), TTFields (tumor treating fields)
      AT A GLANCE COMMENTARY
      Karanam NK, et al.

      Background

      TTFields is a noninvasive physical modality of cancer therapy for the treatment of recurrent and newly diagnosed GBM and advanced MPM in combination with chemotherapies. TTFields mechanism of action has been descried as primarily through the disruption of mitosis, though it is not fully understood yet.

      Translational Significance

      Our study data suggest that there are other opportunities to develop more effective therapeutic strategies using combination therapies that exploit the molecular environment generated by TTFields. Based upon these results we suggest the use of TTFields prior to or concomitant with fractionated radiotherapy and/or chemotherapy agents that either cause DNA damage or replication stress.

      INTRODUCTION

      Tumor treating fields (TTFields) are low-intensity, intermediate frequency, and alternating electric fields that are applied over regions of the body where tumors are localized using noninvasive arrays. The FDA (Food and Drug Administration) has approved Optune (NovoCure Ltd.), a TTFields portable delivery system for the treatment of recurrent and newly diagnosed glioblastoma (GBM) in combination with temozolomide
      • Stupp R.
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      • Kanner A.A.
      • et al.
      Maintenance therapy with tumor-treating fields plus temozolomide vs temozolomide alone for glioblastoma: a randomized clinical trial.
      and for unresectable, locally advanced or metastatic, malignant pleural mesothelioma. Clinical trials are ongoing for other cancers, including lung, pancreatic, and ovarian cancers.
      TTFields are thought to preferentially target cancer cells by exploiting their higher mitotic index and disrupting mitosis through the creation of a nonuniform electric field that induces the dielectrophoretic movement of polar molecules towards the region of higher field intensity, effectively preventing microtubule polymerization and other critical biochemical functions involved in mitosis.
      • Gonzalez C.F.
      • Remcho V.T.
      Harnessing dielectric forces for separations of cells, fine particles and macromolecules.
      • Kirson E.D.
      • Dbaly V.
      • Tovarys F.
      • et al.
      Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors.
      • Kirson E.D.
      • Gurvich Z.
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      • et al.
      Disruption of cancer cell replication by alternating electric fields.
      Furthermore, by adapting the frequency and field intensity to the presumed size and physical properties of dividing tumor cells, nondividing normal cells are spared.
      • Stupp R.
      • Taillibert S.
      • Kanner A.A.
      • et al.
      Maintenance therapy with tumor-treating fields plus temozolomide vs temozolomide alone for glioblastoma: a randomized clinical trial.
      ,
      • Kirson E.D.
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      • Dbaly V.
      • et al.
      Chemotherapeutic treatment efficacy and sensitivity are increased by adjuvant alternating electric fields (TTFields).
      ,
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      • et al.
      Selective toxicity of tumor treating fields to melanoma: an in vitro and in vivo study.
      Preclinical studies have shown additive to synergistic effects when TTFields were used in combination with several classes of chemotherapeutic drugs, such as paclitaxel, cisplatin, pemetrexed, gemcitabine, doxorubicin, 5-FU (Fluorouracil), cyclophosphamide, DTIC (Dacarbazine), Irinotecan, and spindle assembly checkpoint inhibitors.
      • Giladi M.
      • Schneiderman R.S.
      • Porat Y.
      • et al.
      Mitotic disruption and reduced clonogenicity of pancreatic cancer cells in vitro and in vivo by tumor treating fields.
      • Giladi M.
      • Lee S.Y.
      • Hiller R.
      • Chung K.Y.
      • Khananshvili D.
      Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins.
      • Giladi M.
      • Weinberg U.
      • Schneiderman R.S.
      • et al.
      Alternating electric fields (tumor-treating fields therapy) can improve chemotherapy treatment efficacy in non-small cell lung cancer both in vitro and in vivo.
      • Kessler A.F.
      • Frombling G.E.
      • Gross F.
      • et al.
      Effects of tumor treating fields (TTFields) on glioblastoma cells are augmented by mitotic checkpoint inhibition.
      • Voloshin T.
      • Munster M.
      • Blatt R.
      • et al.
      Alternating electric fields (TTFields) in combination with paclitaxel are therapeutically effective against ovarian cancer cells in vitro and in vivo.
      • Schneiderman R.S.
      • Shmueli E.
      • Kirson E.D.
      • Palti Y.
      TTFields alone and in combination with chemotherapeutic agents effectively reduce the viability of MDR cell sub-lines that over-express ABC transporters.
      TTFields also target the calcium channel Cav1.2, and combinations of calcium antagonists, such as benidipine enhance glioma cell killing.
      • Neuhaus E.
      • Zirjacks L.
      • Ganser K.
      • et al.
      Alternating electric fields (TTFields) activate Cav1.2 channels in human glioblastoma cells.
      Furthermore, AMPK-dependent autophagy, which occurred after cells had undergone mitosis in response to aneuploidy and ER (Endoplasmic reticulum) stress, was identified as a survival mechanism upon TTFields exposure and serves as a druggable resistance mechanism to TTFields.
      • Shteingauz A.
      • Porat Y.
      • Voloshin T.
      • et al.
      AMPK-dependent autophagy upregulation serves as a survival mechanism in response to Tumor Treating Fields (TTFields).
      These preclinical results cannot be explained by the interruption of mitosis, which suggests that there are other mechanisms by which TTFields used in combination with different chemotherapeutic agents elicit additive or synergistic cell killing.
      In a previous study that was designed to determine whether there were differences in the response to TTFields in a series of non–small-cell lung cancers (NSCLC) of different genetic backgrounds and radiosensitivity, a genomics approach identified clear differences in gene expression patterns in NSCLC cells that were very sensitive to TTFields exposure compared to cells less responsive to TTFields exposure. A key finding was the decreased expression of the Fanconi's Anemia (FA) pathway genes, which play a key role in DNA double-strand break (DSB) repair
      • Karanam N.K.
      • Srinivasan K.
      • Ding L.
      • Sishc B.
      • Saha D.
      • Story M.D.
      Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.
      upon TTFields exposure. This downregulation of FA genes was accompanied by slower DNA repair postradiation and a greater number of residual (unrepaired) DNA lesions at 48 hours. This was confirmed by Giladi et al., who also described a synergistic enhancement of efficacy when TTFields was used in combination with ionizing radiation (IR) in glioma cells
      • Giladi M.
      • Munster M.
      • Schneiderman R.S.
      • et al.
      Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells.
      including the impairment of radiation- or chemically-induced DNA damage repair. However, while monitoring DSB repair kinetics after IR followed immediately by TTFields exposure, it was noted that the number of γ-H2AX foci was substantially greater than that of γ-H2AX-53BP1 co-localized foci. More interesting was the increase in γ-H2AX foci with time in cells that were not irradiated but were exposed to TTFields. Because γ−H2AX is also an early sensor of stalled replication forks during replication stress,
      • Sirbu B.M.
      • Couch F.B.
      • Feigerle J.T.
      • Bhaskara S.
      • Hiebert S.W.
      • Cortez D.
      Analysis of protein dynamics at active, stalled, and collapsed replication forks.
      it seemed apparent that TTFields exposure was inducing replication stress, as reduced FA pathway gene expression negatively affects the repair of collapsed or stalled replication forks
      • Schlacher K.
      • Wu H.
      • Jasin M.
      A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.
      and the MCM6 and MCM10 genes, integral members of the DNA replication complex, were also downregulated. Indeed, TTFields exposure did induce replication stress as shown by a decrease in replication fork speed and an increased appearance of R-loop formation with time of exposure to TTFields. Based upon these results demonstrating increased DNA damage, replication stress and decreased DNA damage repair capacity as a result of TTFields exposure, that TTFields could prompt the development of a conditional vulnerability in cancer cells, rendering them more sensitive to DNA damaging agents or to agents that increase replication stress. Indeed, the results described herein show that applying TTFields before radiation exposure enhanced radiosensitivity far more than applying TTFields after radiation. Cell killing from cisplatin exposure applied concomitantly with TTFields was synergistic as compared to cell killing by cisplatin or TTFields alone. Inhibition of PARP1, which has a role in DNA repair processes as well as protects DNA from MRE11 degradation during replication fork stalling, in combination with either TTFields alone or with TTFields and radiation applied at the end of TTFields exposure was also synergistic as compared to either modality alone or in combination with TTFields. Finally, gene expression data from NSCLC cells lines collected as a function of time of TTFields exposure indicated that TTFields likely induces DNA damage/replication stress independent of mitotic aberrations, which underscores the important contribution of the DNA damage/replication stress pathway to TTFields’ biological effect.

      MATERIALS AND METHODS

      Cell culture

      All cell lines were grown in RPMI medium
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      • Sato M.
      • Story M.D.
      • et al.
      Non-small-cell lung cancers with kinase domain mutations in the epidermal growth factor receptor are sensitive to ionizing radiation.
      ,
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      • et al.
      Epidermal growth factor and hypoxia-induced expression of CXC chemokine receptor 4 on non-small cell lung cancer cells is regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signaling pathway and activation of hypoxia inducible factor-1alpha.
      supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals) and penicillin/streptavidin (final concentration 50 μg/ml) (Sigma-Aldrich). All cells were grown at 37°C in a humidified incubator constantly supplied with 5% CO2.

      Tumor treatment fields

      TTFields were generated using the inovitro system (NovoCure Ltd.), which uses 2 pairs of electrodes printed perpendicularly on the outer walls of a Petri dish composed of high dielectric constant ceramic (lead magnesium niobate-lead titanite [PMN-PT]). The transducer arrays were connected to a sinusoidal waveform generator that generates low-intensity electric fields at the desired frequencies in the medium, as summarized in supplementary Table 1. The orientation of the TTFields was rotated 90o every 1 second, thus covering most of the orientation axes of cell division, as described by Kirson et al.
      • Kirson E.D.
      • Gurvich Z.
      • Schneiderman R.
      • et al.
      Disruption of cancer cell replication by alternating electric fields.
      Plate temperature was maintained at 37°C by placing the plates in a refrigerated incubator where the temperature was maintained at 19°C to dissipate the heat generated by the inovitro system. The temperature was measured by 2 thermistors (Omega Engineering) attached to the ceramic walls. All cells were grown on a cover slip inside the inovitro dish (NovoCure Ltd.) and treated with TTFields at the times indicated in the figure legends.

      Cell growth assay

      Human NSCLC (H157, H4006, A549, and H1299) cell lines were treated with the chemotherapeutic agents cisplatin (Selleckchem) and olaparib (Selleckchem) at different concentrations for 24, 48, and 72 hours, and cell growth was determined using a Beckman Coulter counter (Beckman Coulter, Inc.) in triplicate for each sample. Growth curves were drawn using the average cell number counted at each time point and the given concentrations using GraphPad Prism V.6 (GraphPad Software, Inc.).

      DNA fiber assay

      Approximately 2.5 × 105 H157 and H1299 cells which were treated or not with TTFields for 15, 30, 45, and 60 hours were labeled with Idoxyuridine (IdU) (Sigma-Aldrich) at a final concentration of 100 μM for 30 minutes while the same number of cells were unlabeled. Then, cells were washed 4 times with warm Phosphate buffered saline (PBS), and both labeled and unlabeled cells were trypsinized and mixed at a 1:15 ratio (labeled:unlabeled). These mixed cells were lysed on a clean glass slide in 25 μl of lysis buffer (0.5% Sodium dodecyl sulfate-SDS, 50 mM Ethylenediaminetetraacetic acid-EDTA, and 200 mM Tris-HCl pH 7.4) for 8 minutes; slides were tilted slightly at a ∼15o angle to help DNA spread slowly. After air drying the samples, DNA was fixed in methanol:acetic acid (3:1) at room temperature for 8–10 minutes and allowed it to air dry. Slides were either kept in 70% ethanol at 4°C until use or advanced directly to immunostaining. For immunostaining, slides were first washed 3 times with PBS at room temperature. To denature the DNA, slides were incubated in 2.5 N HCl in a glass jar at 37°C for 50 minutes, then neutralized them by washing them 4 times with PBS. Following neutralization, slides were incubated in PBS containing 5% goat serum for 2 hours at room temperature. Mouse anti–BrdU antibody was diluted in 5% goat serum, 0.1% Triton X-100 in PBS and incubated at 37°C for 1 hour in a humidified chamber. Then, slides were washed 3 times in PBS containing 0.1% Triton X-100 (PBST) and incubated with antimouse Alexa 488 secondary antibody (Life Technologies) in PBS containing 5% goat serum, 0.1% Triton X-100 for an additional 1 hour. Then, slides were mounted in mounting medium without DAPI (Vector Laboratories Inc.). Images were acquired using a fluorescent microscope (Axio Imager M2, Carl Zeiss) with a 63 × objective (oil immersion, aperture 1.3), and DNA fiber lengths were measured using Axiovision Software (Carl Zeiss).

      Quantification of R-loops using dot blot

      H157 and H1299 cells—which were treated or not with TTFields for 24, 48 and 72 hours—were harvested, and total nucleic acid extraction was performed using the DNeasy blood and tissue kit (Qiagen) according to manufacturer's instructions. The concentration of extracted nucleic acid was measured using a NanoDrop 2000 (Thermo Scientific) and 1.5 μg nucleic acid per condition per replicate was divided into 3 equal portions (500 ng each). Two portions were used for R-loop detection, and the third portion was used for ssDNA detection, which serves as a loading control. One of the 2 portions used for R-loop detection was treated with RNase H (Invitrogen) overnight, which serves as a negative control. The loading control portion of the DNA was denatured for 10 minutes in 0.5 N NaOH, 1.5 M NaCl and neutralized for another 10 minutes in 1M NaCl, 0.5 M Tris-HCl pH7. These 3 samples were loaded onto a positively charged nylon membrane Immobilon-NY+ (Millipore) using a dot-blot apparatus (Biorad). UV crosslinking (0.12 J/m2) and blocking were performed before the membrane portion for R-loop detection was incubated with S9.6 antibody (Kerafast – dilution 1:1000), whereas the loading portion of the membrane was incubated with ssDNA antibody (Sigma-Aldrich – dilution 1:10,000) in PBST containing 2% bovine serum albumin (Thermo Fisher Scientific Inc) overnight at 4°C. The next day, membranes were washed 3 times for 5 minutes each and incubated with their respective secondary antibodies conjugated with horseradish peroxidase (GE Healthcare) for 1 hour at room temperature. After incubating with the secondary antibody, membranes were washed thoroughly with PBST. Membranes were developed using a chemiluminescence detection kit (Thermo Fisher Scientific Inc) on a FluorChem M system (ProteinSimple). Quantification was performed using ImageJ software (NIH) and normalized using the corresponding ssDNA loading control density.

      Immunofluorescence detection of R-loops

      Cells were seeded on glass coverslips and, after treatment, washed and fixed with ice cold methanol. The samples were blocked with 10% normal goat serum for 1 hour and incubated with S9.6a antibody (Kerafast). Samples were washed 3 times for 5 minutes in PBS, then incubated with Alexa Fluor 488-conjugated anti-rabbit antibody (Life Technologies) and Alexa Fluor 555-conjugated anti-mouse antibody (Invitrogen) for 1 hour. Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) contained in Vecatshield mounting medium (Vector Laboratories Inc.). The stained cells were then analyzed under a fluorescence microscope (Axio Imager M2, Carl Zeiss) with a 63 × objective (oil immersion, aperture 1.3) with 5 slices of z-stacks of 0.2 µM thickness each. Quantitative image analysis of 50 nuclei from each experiment was performed manually.

      Quantitative real-time polymerase chain reaction (qRT-PCR)

      Total RNA was extracted using the TRIzol reagent, per protocol (Invitrogen) and resuspended in nuclease-free water (Thermo Scientific). cDNA was synthesized using Applied Biosystems High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR amplification was performed using the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad), with primer sets designed using Primer3 (v.0.4.0) software
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      • Koressaar T.
      • et al.
      Primer3–new capabilities and interfaces.
      and synthesized by Thermo Fisher Scientific (Supplementary File 1). PowerUp SYBR Green Master Mix (Applied Biosystems) was utilized for real-time PCR amplification. The data were analyzed using the 2−ΔΔCt method,
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.
      using the GAPDH mRNA as an endogenous control.

      Clonogenic cell survival assay after treating with chemotherapeutic agents and radiation

      To examine the effect of radiation sensitivity on NSCLCs, exponentially growing cells were treated with IR using a Mark II 137Cs irradiator (JL Shepherd and Associates) at a dose rate of 3.47 Gy/min immediately after application of TTFields for 24, 48, and 72 hours. Cells were treated with chemotherapeutic agents such as cisplatin (Selleckchem) and olaparib (Selleckchem) at concentrations mentioned once together with beginning of TTFields treatment. Cells were then reseeded into 60 mm dishes and incubated for up to 2 weeks. Colonies containing 50 or more cells were considered viable. The data are presented as the mean ± SEM of 3 independent experiments. The enhanced radiosensitivity seen with TTFields was evaluated according to the Highest Single Agent (HSA)
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      • Geary N.
      Understanding synergy.
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      • Zimmermann G.R.
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      • et al.
      Chemical combination effects predict connectivity in biological systems.
      approach by calculating the combination index (CI) as given below:
      CI(AxB)=(SFAXSFB)/SFA+BCI(AxBxC)=(SFAXSFBXSFC)/SFA+B+C


      where SF = Surviving fraction.
      Survivingfraction=NumbersofcoloniesformedNumbersofcoloniesseeded×platingefficiency


      The combination effect was considered enhanced or synergistic when the CI > 1, and additive when the CI = 1. Statistical significance for a positive effect was determined by the p value of a 2-way ANOVA multiple comparison statistical test comparing the combination (TTFields plus IR plus olaparib) to the double agent showing the greatest cell killing for a given dose and time after IR.

      RESULTS

      TTFields treatment decreases replication fork speed and induces replication stress

      An earlier study of DNA damage repair kinetics described higher levels of γ-H2AX foci compared to co-localized γ-H2AX/53BP1 foci and increased chromatid-type aberrations with TTFields exposure,
      • Karanam N.K.
      • Srinivasan K.
      • Ding L.
      • Sishc B.
      • Saha D.
      • Story M.D.
      Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.
      Phosphorylated γ-H2AX has been described as an early sensor of DNA replication stress
      • Sirbu B.M.
      • Couch F.B.
      • Feigerle J.T.
      • Bhaskara S.
      • Hiebert S.W.
      • Cortez D.
      Analysis of protein dynamics at active, stalled, and collapsed replication forks.
      but because phosphorylated γ-H2AX can be generated by different DNA damage types it is not necessarily specific to replication stress. However, as seen in Fig 1, A, the expression of Mini-Chromosome Maintenance 10 (MCM10) and Mini-Chromosome Maintenance 6 (MCM6) genes, which are essential for replication initiation and DNA elongation processes, was decreased with time of exposure to TTFields supporting the notion of replication stress as a consequence of TTFields exposure (Fig 1, A).
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      • Aguilera A.
      Replication stress and cancer.
      ,
      • Looke M.
      • Maloney M.F.
      • Bell S.P.
      Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase.
      Fig 1
      Fig 1TTFields exposure decreases expression of MCM10 and MCM6 genes and replication fork speed over time. A) Normalized gene expression ratio (log2) kinetics of MCM10 and MCM6 genes upon TTFields exposure relative to control in panel of NSCLC cells from micro array results. Newly synthesized DNA length was measured by the DNA fiber assay using halogenated nucleotides analogues in H157 and H1299 cells in 3 separate experiments. Quantification of mean DNA fiber length ± SD at each time point for each cell line is in the table. Histogram graphs showing the frequency distribution of DNA fiber lengths in B) H157 and C) H1299 cells. D) Box plot showing the ranges of DNA fiber lengths at each time point and condition in H157 and H1299 cells were shown as means ± SD from n = 3 experiments. *P< 0.05, **P < 0.01, ***P < 0.001, 2-tailed t test.
      Several reports have implicated the FA pathway proteins in maintaining genome integrity during DNA replication, transcription and replication stress.
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      • Chun J.
      • Powell S.N.
      BRCA1 and BRCA2: different roles in a common pathway of genome protection.
      • Schwab R.A.
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      • Shah F.
      • et al.
      The fanconi anemia pathway maintains genome stability by coordinating replication and transcription.
      • Jones M.J.
      • Huang T.T.
      The Fanconi anemia pathway in replication stress and DNA crosslink repair.
      Replication protein A (RPA) and Chk1 phosphorylation by ATR (Ataxia Telangiectasia and Rad3-related) kinase, or ssDNA detection serve as specific replication stress markers.
      • Zeman M.K.
      • Cimprich K.A.
      Causes and consequences of replication stress.
      RPA protects single-stranded DNA at stalled replication forks
      • Chen R.
      • Wold M.S.
      Replication protein A: single-stranded DNA's first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair.
      and immunofluorescence detection of RPA foci after 72 hours of TTFields exposure identified increased RPA recruitment in H1299 and H157 cells (Supplementary Fig 01).
      Directly measuring polymerase progression using DNA fiber or DNA combing assays, which can track the incorporation of nucleotide analogs, is highly specific and yields the clearest readout of replication stress.
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      • Poli J.
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      • et al.
      Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing.
      Halogenated nucleotides were used to track the length of newly synthesized DNA using a DNA fiber assay. Representative DNA fibers images are shown in Supplementary Fig 2. The length of approximately 50 DNA fibers was measured for each condition and time point. By 30 hours of TTFields exposure, DNA fiber length in both H157 (Fig 1, B and D) and H1299 cells (Fig 1, C and D) was considerably shorter when exposed to TTFields as opposed to cells not exposed to TTFields (19% and 22%, respectively) with the difference in DNA fiber length becoming larger with time of exposure (22% and 34% at 45 hours and 49% and 47% by 60 hours in H157 and H1299 cells, respectively).

      TTFields increases R-loop formation

      R-loops are 3-stranded nucleic acid structures that consist of a DNA-RNA hybrid and a displaced single-stranded DNA. R-loops are dynamically formed during transcription, which can occupy between 5% and 10% of the genome. A head-on collision between the transcription machinery and a replication fork can lead to persistent R-loop formation, which leads to DNA damage and cell death. BRCA1 and BRCA2 function to resolve R-loops, and knockdown of BRCA1 and BRCA2 results in increased R-loops, DNA damage, and replication stress.
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      • Skourti-Stathaki K.
      • Ventz S.
      • et al.
      BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair.
      R-loops were quantified as a function of TTFields exposure time using the R-loop–specific antibody S9.6a to quantify R-loop formation by dot-blot (Fig 2). Denatured single-stranded DNA served as a loading control and RNase H treatment, was used to show the specificity of the S9.6a antibody for DNA-RNA hybrids through the degradation of R-loops (Fig 2, A). TTFields increased R-loop formation with increasing time of TTFields exposure in the more TTFields responsive H157 cell line while in the less responsive H1299 cell line, R-loop formation plateaued at 48 hours. To corroborate the production of R-loops using the dot-blot assay the S9.6a antibody was tagged with an immunofluorescent secondary antibody and R-loop foci formation was determined after 72 hours of TTFields exposure. The mean intensity per nucleus in approximately 50 nuclei per condition was quantified and yielded the same result as the dot-blot assay, that is, that TTFields exposure significantly increased R-loop formation in H157 and H1299 cells (Fig 2, B).
      Fig 2
      Fig 2TTFields treatment facilitates R-loop formation. TTFields exposure increases R-loop formation In H157 and H1299 cells. R-loop formation was quantified by A) dot-blot after different time points of TTFields exposure indicated and B) immunofluorescence after 72 hours of TTFields exposure using a DNA-RNA hybrid-specific S9.6 antibody. Representative images of dot-blots and immunofluorescence are shown here. Quantitative data are shown as means ± SD from n = 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed t test.

      TTFields treatment before IR increases radiation-induced cell killing

      Based on these newly identified inhibitory effects by TTFields’ exposure on the DNA damage response and the replication stress pathways, it was postulated that that TTFields exposure results in a conditional vulnerability to DNA damaging agents such as radiation and cisplatin, or to replication fork maintenance/DNA repair pathway inhibitors such as olaparib (PARP inhibitor). Comparison of clonogenic survival and CI values from the survival data in 2 different experiments: (1) TTFields applied after IR treatment, and (2) TTFields applied before IR treatment showed that NSCLC cells were more susceptible to radiation when they were exposed to TTFields before IR treatment than when they were given IR treatment first (Fig 3). Switching the sequence of TTFields treatment increased the CI in all cell lines irrespective of their relative sensitivity to TTFields alone.
      Fig 3
      Fig 3TTFields treatment before IR increases radiation-induced cell killing. The Combination Index (CI) values for H157 and H1299 cell lines were higher when applying A) TTFields exposure before radiation than B) radiation before TTFields exposure. The combinatorial effect was evaluated using the Highest Single Agent (HSA) approach and is presented in the tables for each condition and time point shown. Clonogenic quantitation data are shown as means ± SD from n = 3 experiments. *P < 0.05, **P < 0.01, 2-tailed t test according to HSA approach.

      TTFields enhance cisplatin sensitivity in NSCLC cells

      To exploit replication stress TTFields was combined with chemotherapeutic agents, which can also cause replication stress at several key steps. Platinum compounds (cisplatin) are known to produce DNA inter- and intrastrand crosslinks between nucleotide bases.
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      The intrastrand crosslinks produce DNA lesions in the template strand, and the interstrand crosslinks lead to defects in DNA unwinding, which is the essential first step of replication.
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      ,
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      Y-family DNA polymerases and their role in tolerance of cellular DNA damage.
      The PIC25 (plating inhibitory concentration for 25%) was determined for 4 NSCLC cell lines and each cell line was treated by its respective PIC25 in combination with TTFields for the times indicated in Fig 4. the CI and p values were calculated using the HSA approach for each cell line are given in table (Fig 4). TTFields synergistically increased the efficacy of cisplatin exposure in all 4 NSCLC cell lines tested, though the degree of synergy varied across cell lines (Fig 4).
      Fig 4
      Fig 4TTFields synergistically enhance cisplatin efficacy. TTFields treatment enhanced cell killing by cisplatin, likely by impairing the repair of DNA crosslinks generated by cisplatin exposure. The table of Combination Index and p values of TTFields and cisplatin combination therapy suggests synergy (CI > 1). The comparison analysis used the Highest Single Agent (HSA) approach. Clonogenic quantitation data are shown as means ± SD from n = 3 experiments. *P < 0.05, **P < 0.01, 2-tailed t test according to HSA approach.

      PARP1 inhibitor cytotoxicity is enhanced when used in combination with TTFields alone or with TTFields and IR

      PARP1 protects DNA breaks by recruiting DNA repair and checkpoint proteins at the sites of damage.
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      • et al.
      Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpoint proteins.
      PARP1 also helps to stabilize stalled replication forks, recruits MRE11 to perform the end-processing required for replication restart, and enhances Chk1 activation.
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      Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.
      Both BRCA1 and BRCA2 play important roles in genome stability via their roles in the DNA damage response, DNA repair and specific to this study, DNA replication fork stabilization.
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      BRCA1-BARD1 promotes RAD51-mediated homologous DNA pairing.
      Under replication stress BRCA2 relocates to stalled replication forks and promotes the formation of stable RAD51 nucleofilaments which protects DNA strands from the MRE11 nuclease thereby suppressing replication fork degradation.
      • Schlacher K.
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      Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains.
      • Ying S.
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      Mre11-dependent degradation of stalled DNA replication forks is prevented by BRCA2 and PARP1.
      BRCA1 also plays a role in fork stabilization pathway through the suppression of MRE11 activity and loss of either BRCA1 or BRCA2 negatively impact replication fork stabilization.
      • Schlacher K.
      • Wu H.
      • Jasin M.
      A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.
      ,
      • Higgs M.R.
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      • et al.
      BOD1L is required to suppress deleterious resection of stressed replication forks.
      ,
      • Kotsantis P.
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      Cancer therapy and replication stress: forks on the road to perdition.
      TTFields reduces the expression of FA pathway genes including BRCA1, FANCM, and FANCD2, all of whom are involved in fork stabilization. PARP1 activation at stalled replication forks recruits MRE11 to stalled forks to mediate fork recovery, however, in cells lacking BRCA1/2, fork degradation is extensive.
      • Chaudhuri A.R.
      • Callen E.
      • Ding X.
      • et al.
      Erratum: replication fork stability confers chemoresistance in BRCA-deficient cells.
      ,
      • Ding X.
      • Ray Chaudhuri A.
      • Callen E.
      • et al.
      Synthetic viability by BRCA2 and PARP1/ARTD1 deficiencies.
      This rationale, along with the notion of synthetic lethality in BRCA1/2 deficient cells treated with PARP inhibitors,
      • Hartwell L.H.
      • Szankasi P.
      • Roberts C.J.
      • Murray A.W.
      • Friend S.H.
      Integrating genetic approaches into the discovery of anticancer drugs.
      ,
      • Helleday T.
      The underlying mechanism for the PARP and BRCA synthetic lethality: clearing up the misunderstandings.
      led to the presumption that PARP1 inhibitors together with TTFields would further increase replication stress and be synergistically lethal. Proliferation assays using different concentrations of olaparib and clonogenic survival assays using different concentrations of olaparib in combination with TTFields were performed to determine an optimal concentration of olaparib to be used in further experiments (Supplementary Fig 3). A 10 μM concentration of olaparib for which a moderate cell killing effect was observed was used in combination experiments. Clonogenic assays were carried out using olaparib concomitantly with TTFields for increasing time and concomitantly with TTFields followed by radiation at the end of the TTFields exposure time (Fig 5). There was synergistic cell killing when TTFields was used in combination with the PARP1 inhibitor olaparib with or without IR suggesting that TTFields exposure does cause vulnerability to multiple agents that damage DNA and/or interfere with replication fork maintenance; however, the highest synergy based upon CI values was seen with the triple combination of TTFields, olaparib, and then radiation.
      Fig 5
      Fig 5TTFields synergistically enhance the cytotoxicity of the PARP inhibitor olaparib which is further enhanced when IR is added to the treatment regime. The combination therapies of TTFields plus olaparib and TTFields plus radiation decreases the clonogenic survival of H157 and H1299 cells. The triple combination, TTFields plus olaparib plus radiation, further increased the Combination Index. Combination effects were evaluated using a Highest Single Agent (HSA) approach for different combinations, as shown in the table. Combination index > 1 and p value < 0.05 denote a synergistic effect for a given time point. Clonogenic quantitation data are shown as means ± SD from n = 3 experiments. *P < 0.05, **P < 0.01, 2-tailed t test according to HSA approach.

      TTFields-induced replication stress is an effector and occurs in parallel with mitotic aberrations

      Having identified replication stress as a new mechanism by which TTFields kills tumor cells in addition to the disruption of mitosis, one could ask whether these processes are actually independent of one another. For example, assuming TTFields exposure increases replication stress, does the replication stress induced DNA damage caused by TTFields exposure drive mitotic aberrations or do the mitotic aberrations generated during mitosis drive replication stress in cells that are able to slip through mitosis?
      Pedersen et al
      • R S.P.
      • Karemore G.
      • Gudjonsson T.
      • et al.
      Profiling DNA damage response following mitotic perturbations.
      systematically examined the crosstalk between chromosome separation in mitosis and DNA replication during S phase as major sources of intrinsic genome instability. A mitocheck library of 1249 genes was tested using siRNA screening followed by live cell imaging. Forty-seven genes were identified as key upstream regulators that directly cause mitotic delays. Mitotic aberrations and DNA damage response (DDR) markers were monitored together with mitotic delays and these 47 genes were divided into 3 sub-groups: genes that cause (1) mild mitotic aberrations with little or no evidence of DDR, (2) mitotic aberrations before any evidence of DDR, and (3) mitotic aberrations after evidence of DDR. TTFields-induced gene expression array data was interrogated to see how TTFields exposure affected the expression of these 47 genes. TTFields reduced the expression of 33 out of 47 genes (∼70%) of these key regulator genes in all 4 cell lines, which mainly belong to 2 major sub-groups (mitotic aberrations before evidence of DDR and mitotic aberrations after evidence of DDR), mimicking the siRNA silencing as shown in Pedersen et al’s study (Fig 6A). The expression of these genes was often reduced as early as 24 hours and further so by 48 hours (eg, PLK1, KIF23, WEE1, PAFAH1, and SNRNP200); again mimicking the observations made in Pedersen et al. As the kinetics for these different processes are very similar, it is likely that the production of mitotic events and replication stress occur simultaneously, that is, independent of one another. To further validate the decreased expression of these key regulators the relative expression of 2 genes from each group of mitotic aberrations before evidence of DDR (KIF11 and CENPE) and mitotic aberrations after evidence of DDR (WEE1 and SNRNP200) was quantified and compared to respective controls in H157 (more TTFields sensitive) and H1299 (less TTFields sensitive) cells. Gene specific primers (Supplementary File 1) were used to quantify using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and GAPDH expression was used to normalize the expression values across the conditions. qRT-PCR data suggest that the expression of all 4 genes expression was decreased with TTFields exposure time in both cell lines tested (Fig 6, B and C)
      Fig 6
      Fig 6Temporal gene expression results point to reduced expression of key regulators of mitosis and replication stress genes under TTFields treatment condition. A) Genes were divided into 3 subgroups based on their functional outcome whether mitotic aberrations occur with no or little DDR or mitotic aberrations occur before DDR or mitotic aberrations occur after DDR. The heat map of temporal gene expression changes for these 47 key regulatory genes suggest that TTFields reduces the expression of most of these genes. Decreased expression of 2 genes from groups of mitotic aberrations before DDR (KIF11 and CENPE) and after DDR (WEE1 and SNRNP200) was verified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in B) H157, C) H1299 cells and are shown as means of log2 expression change ± SD from n = 3 experiments after 24 hours and 48 hours of TTFields exposure compare to respective controls.

      DISCUSSION

      The DNA replication and transcription processes are crucial for the completion of the cell cycle and for cell viability, and function simultaneously on the same DNA template. The transcription machinery of each gene, depending on its coding strand, moves either head-on (lagging strand genes) or co-directionally (leading strand genes) relative to the movement of the replication machinery (replisome).
      • Lang K.S.
      • Hall A.N.
      • Merrikh C.N.
      • et al.
      Replication-transcription conflicts generate R-loops that orchestrate bacterial stress survival and pathogenesis.
      Co-directional encounters between transcription and replication machineries lead to stalled replication and a restart of the replication fork
      • Merrikh H.
      • Machon C.
      • Grainger W.H.
      • Grossman A.D.
      • Soultanas P.
      Co-directional replication-transcription conflicts lead to replication restart.
      and sometimes to DNA breaks.
      • Dutta D.
      • Shatalin K.
      • Epshtein V.
      • Gottesman M.E.
      • Nudler E.
      Linking RNA polymerase backtracking to genome instability in E. coli.
      Head-on encounters are more detrimental and persistent, and they promote genomic instability.
      • Million-Weaver S.
      • Samadpour A.N.
      • Merrikh H.
      Replication restart after replication-transcription conflicts requires RecA in Bacillus subtilis.
      • Million-Weaver S.
      • Samadpour A.N.
      • Moreno-Habel D.A.
      • et al.
      An underlying mechanism for the increased mutagenesis of lagging-strand genes in Bacillus subtilis.
      • Merrikh C.N.
      • Brewer B.J.
      • Merrikh H.
      The B. subtilis accessory helicase PcrA facilitates DNA replication through transcription units.
      Evolutionarily highly transcribed and essential genes are usually expressed co-directionally to minimize head-on collisions.
      • Rocha E.P.
      • Danchin A.
      Essentiality, not expressiveness, drives gene-strand bias in bacteria.
      However, many stress-responsive and pathogenic genes are expressed head-on.
      • Nicolas P.
      • Mader U.
      • Dervyn E.
      • et al.
      Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis.
      ,
      • Paul S.
      • Million-Weaver S.
      • Chattopadhyay S.
      • Sokurenko E.
      • Merrikh H.
      Accelerated gene evolution through replication-transcription conflicts.
      The clearest and most direct readout of replication stress comes from directly measuring polymerase progression using a DNA fiber assay. Thus, using nucleotide analogs, newly synthesized DNA length was measured and confirmed that TTFields decreases the speed of DNA replication (Fig 1, B–D), which suggests that TTFields induces replication stress. Although these results do not distinguish between co-directional and head-on collisions, it is clear that under TTFields treatment, these collisions are not resolved properly to facilitate the proper progression of replication given reduced expression of the FA pathway
      • Karanam N.K.
      • Srinivasan K.
      • Ding L.
      • Sishc B.
      • Saha D.
      • Story M.D.
      Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.
      and MCM genes (Fig 1, A) upon TTFields exposure.
      • Karanam N.K.
      • Srinivasan K.
      • Ding L.
      • Sishc B.
      • Saha D.
      • Story M.D.
      Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.
      ,
      • Gaillard H.
      • Garcia-Muse T.
      • Aguilera A.
      Replication stress and cancer.
      ,
      • Looke M.
      • Maloney M.F.
      • Bell S.P.
      Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase.
      Changes in DNA topology during collisions between replication and transcription machineries cause DNA-RNA hybrid structures called R-loops to form. R-loops have been implicated in a multitude of physiological roles in cells, such as regulation of gene expression, replication, Ig class switch recombination, DNA repair, and genomic instability.
      • Chan Y.A.
      • Aristizabal M.J.
      • Lu P.Y.
      • et al.
      Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.
      • El Hage A.
      • Webb S.
      • Kerr A.
      • Tollervey D.
      Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria.
      • Ginno P.A.
      • Lott P.L.
      • Christensen H.C.
      • Korf I.
      • Chedin F.
      R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters.
      • Stirling P.C.
      • Chan Y.A.
      • Minaker S.W.
      • et al.
      R-loop-mediated genome instability in mRNA cleavage and polyadenylation mutants.
      Recent findings highlight the contribution of defective R-loop control to several human diseases
      • Richard P.
      • Manley J.L.
      R loops and links to human disease.
      ,
      • Sollier J.
      • Cimprich K.A.
      Breaking bad: R-loops and genome integrity.
      and it is clear that TTFields exposure increases R-loop formation (Fig 2) as a function of time in keeping with the notion of increased replication stress.
      Given that TTFields exposure results in reduced expression of specific DNA repair, replication fork, chromosome maintenance, and cell cycle regulatory genes, combining agents that target replication fork maintenance processes or DNA repair may be more lethal when combined with TTFields. This would be particularly true when TTFields is applied before, or at the very least concomitant with, such agents. This was the case when NSCLC cells were found to be more susceptible to radiation when they were exposed to TTFields before IR treatment compared to their response to radiation given before TTFields (Fig 3), and when TTFields was applied concomitantly with cisplatin (Fig 4). Furthermore, the combination of TTFields and the PARP1 inhibitor olaparib followed by radiation at the end of TTFields exposure was synergistic compared to radiation or olaparib alone or in combination (Fig 5). By altering the scheduling of combinations synergy was achieved in nearly all cases as defined by the HSA approach and statistically significant CI values. One might proceed with caution with the use of PARP1 inhibitors given that depletion of PARP1 prior to replication stalling has been shown to increase cell viability in BRCA2-deficient cells
      • Chaudhuri A.R.
      • Callen E.
      • Ding X.
      • et al.
      Erratum: replication fork stability confers chemoresistance in BRCA-deficient cells.
      ,
      • Ding X.
      • Ray Chaudhuri A.
      • Callen E.
      • et al.
      Synthetic viability by BRCA2 and PARP1/ARTD1 deficiencies.
      ,
      • Mijic S.
      • Zellweger R.
      • Chappidi N.
      • et al.
      Replication fork reversal triggers fork degradation in BRCA2-defective cells.
      and cells resistant to PARP1 inhibition are also tolerant of cisplatin and topotecan.
      • Chaudhuri A.R.
      • Callen E.
      • Ding X.
      • et al.
      Erratum: replication fork stability confers chemoresistance in BRCA-deficient cells.
      This suggests that stalled replication fork stabilization confers resistance to replication stress-inducing chemotherapeutics but because of the general suppression of a host of genes and proteins involved in replication fork maintenance and stability, checkpoint control and DNA repair pathways, TTFields exposure may overcome such resistance. Indeed the results herein provide a rationale for the additive to synergistic effects of TTFields seen in earlier preclinical reports
      • Kessler A.F.
      • Frombling G.E.
      • Gross F.
      • et al.
      Effects of tumor treating fields (TTFields) on glioblastoma cells are augmented by mitotic checkpoint inhibition.
      ,
      • Schneiderman R.S.
      • Shmueli E.
      • Kirson E.D.
      • Palti Y.
      TTFields alone and in combination with chemotherapeutic agents effectively reduce the viability of MDR cell sub-lines that over-express ABC transporters.
      • Neuhaus E.
      • Zirjacks L.
      • Ganser K.
      • et al.
      Alternating electric fields (TTFields) activate Cav1.2 channels in human glioblastoma cells.
      • Shteingauz A.
      • Porat Y.
      • Voloshin T.
      • et al.
      AMPK-dependent autophagy upregulation serves as a survival mechanism in response to Tumor Treating Fields (TTFields).
      • Karanam N.K.
      • Srinivasan K.
      • Ding L.
      • Sishc B.
      • Saha D.
      • Story M.D.
      Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.
      with chemotherapeutic drugs such as paclitaxel, cisplatin, gemcitabine, doxorubicin, 5-FU, cyclophosphamide, DTIC, and Irinotecan which are known to primarily increase replication stress but not directly affect mitosis.
      • Giladi M.
      • Schneiderman R.S.
      • Porat Y.
      • et al.
      Mitotic disruption and reduced clonogenicity of pancreatic cancer cells in vitro and in vivo by tumor treating fields.
      • Giladi M.
      • Lee S.Y.
      • Hiller R.
      • Chung K.Y.
      • Khananshvili D.
      Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins.
      • Giladi M.
      • Weinberg U.
      • Schneiderman R.S.
      • et al.
      Alternating electric fields (tumor-treating fields therapy) can improve chemotherapy treatment efficacy in non-small cell lung cancer both in vitro and in vivo.
      • Kessler A.F.
      • Frombling G.E.
      • Gross F.
      • et al.
      Effects of tumor treating fields (TTFields) on glioblastoma cells are augmented by mitotic checkpoint inhibition.
      • Voloshin T.
      • Munster M.
      • Blatt R.
      • et al.
      Alternating electric fields (TTFields) in combination with paclitaxel are therapeutically effective against ovarian cancer cells in vitro and in vivo.
      DNA replication is a mandatory and prerequisite step for mitosis. Both replication and mitosis are tightly coupled, regulated by feedback mechanisms and are major independent regulators of genome integrity. Several reports indicate that TTFields induce mitotic aberrations, and it is reported here that TTFields exposure induces replication stress and increased DNA damage as measured by the appearance of DNA repair foci. Whether these events are independent or linked is unknown nor is it known to what extent each event contributes to the full effect of TTFields exposure. Pederson et al. described 3 temporal profiles for DNA repair and mitotic aberrations: (1) mild mitotic aberrations with little or no evidence of DDR; (2) mitotic aberrations appearing before evidence of DDR; and (3) mitotic aberrations after evidence of DDR based upon the knockout of 47 genes that impact these events. The gene expression data from TTFields exposures of up to 48 hours in the 4 NSCLC cell lines described here were mapped onto the 47 genes described by Pederson (Fig 6). The expression of genes in the sub-groups of mitotic aberrations before DDR and after DDR were reduced at 24 hours post-TTFields exposure and further reduced after 48 hours of TTFields exposure. These time points correlate with time points where TTFields induced cell killing, decreased replication fork speed and increased R-loop formation occurred (data from current study) and with increased mitotic aberrations and DNA damage as seen by others.
      • Giladi M.
      • Lee S.Y.
      • Hiller R.
      • Chung K.Y.
      • Khananshvili D.
      Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins.
      ,
      • Karanam N.K.
      • Srinivasan K.
      • Ding L.
      • Sishc B.
      • Saha D.
      • Story M.D.
      Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.
      ,
      • Kim E.H.
      • Kim Y.J.
      • Song H.S.
      • et al.
      Biological effect of an alternating electric field on cell proliferation and synergistic antimitotic effect in combination with ionizing radiation.
      The gene expression patterns described in Fig 6 suggest that for an asynchronously growing population of tumor cells, the mitotic aberrations induced by TTFields and the DNA damage induced by replication stress can occur independently with both contributing to TTFields’ ultimate biological effect. However, because there are intermediate structures generated during replication stress that do not appear as DNA damage until a cell reaches mitosis, and, there are mitotic aberrations that appear in the subsequent interphase (marked by DNA repair foci), it suggests that these processes are also intimately linked (Fig 7). As to the extent to which contributes to cell fate, it could be argued that the simple ratio of cells in S phase vs cells in mitosis points to S phase events contributing more to cell fate. Those S phase events would appear as frank DNA damage events that are not repaired and which could prevent a cell from reaching mitosis, or would appear in mitosis as either chromosomal aberrations or as a mitotic catastrophe event. The more modest number of cells in mitosis for which aberrations are generated via errors in karyokinesis could lead to mitotic catastrophe or would appear as damaged DNA in the next interphase and subsequently die by apoptosis (Fig 7).
      Fig 7
      Fig 7Diagrammatic representation of summary of TTFields major mechanisms of action. TTFields exposure increases mislocalization of septins and mitotic spindle disruption which causes abnormal cell division and chromosome segregation leading to mitotic catastrophe and cell death. TTFields exposure decreases the expression of FA pathway genes which are implicated in DNA damage repair and replication fork stabilization processes. Under higher replication stress conditions in the presence of TTFields and lower levels of FA pathway players failure of replication protection serves as endogenous source of DNA damage. A poor response to ongoing high replication stress and DNA damage repair process under TTFields exposure leads to decreased replication fork speed and increased replication errors, R-loops formation and genomic instability which eventually lead to cell death. The cell cycle is tightly regulated through several checkpoint feedback mechanisms in which TTFields induced mitotic aberrations in M-phase and increased replication stress in S-phase contribute at least in part independently and simultaneously towards TTFields induced cell death.
      In summary, TTFields exposure causes systemic perturbations to key molecular and biochemical pathways (Fig 7) that render tumor cells vulnerable to a number of different agents. A limitation to these studies is the lack of translation to the in vivo condition. Currently, it is not possible to generate such data as the mechanism by which in vivo experiments can be performed is not yet available. Once such is available, in vivo validation experiments can be performed. However, the results herein could be considered as an explanation for clinical trial results where replication stress inducing agents were combined with TTFields.
      • Ceresoli G.
      • Aerts J.
      • Madrzak J.
      • et al.
      MA12.06 STELLAR – final results of a phase 2 trial of TTFields with chemotherapy for first-line treatment of malignant pleural mesothelioma.
      ,
      • Lu G.
      • Rao M.
      • Zhu P.
      • et al.
      Triple-drug therapy with bevacizumab, irinotecan, and temozolomide plus tumor treating fields for recurrent glioblastoma: a retrospective study.
      Finally, TTFields are now applied either alone, postsurgery and/or radiation, and often concomitantly with chemotherapy agents. The data herein suggest that other therapeutic strategies should be considered so as to take advantage of the vulnerabilities generated by prior or concomitant TTFields exposure. Possible alternative strategies could include using TTFields as a neoadjuvant therapy. Furthermore, TTFields could be used during a patient's course of radiotherapy or radio-chemotherapy. The implications for cancer therapy could be profound.

      ACKNOWLEDGMENTS

      A Sponsored Research Agreement between MDS and Novocure provided funding for these studies. We thank Dr. Jonathan Feinberg of the Department of Radiation Oncology, UT Southwestern Medical Center for editorial support. All authors have read the journal's authorship agreement and the manuscript has been reviewed by and approved by all named authors.
      Funding: Funding provided by NovoCure Ltd, to MDS.
      Conflicts of Interest: All authors have read the journal's policy on disclosure of potential conflict of interest. NKK and MDS are inventors on US patent application titled “Treating tumors using Tumor Treating Fields combined with a PARP inhibitor” which is pending approval. The other authors declare that they have no other competing interests.
      Author contributions: Conception and design: Narasimha Kumar Karanam, and Michael Story; Acquisition of data: Narasimha Kumar Karanam, and Lianghao-Ding; Analysis and interpretation of the data: all authors; Drafting the article: Narasimha Kumar Karanam; Critically revising the article: all authors; Study supervision: Michael Story
      Data statement: All data supporting the conclusion of this manuscript are included as main and supplementary materials.

      Appendix. Supplementary materials

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