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. 2021 Mar 1:500:220-227.
doi: 10.1016/j.canlet.2020.12.023. Epub 2020 Dec 21.

Targeting NPM1 in irradiated cells inhibits NPM1 binding to RAD51, RAD51 foci formation and radiosensitizes NSCLC

Geri Traver  1 , Konjeti R Sekhar  1 , Peter A Crooks  2 , Diane S Keeney  3 , Michael L Freeman  4
Affiliations

Targeting NPM1 in irradiated cells inhibits NPM1 binding to RAD51, RAD51 foci formation and radiosensitizes NSCLC

Geri Traver et al. Cancer Lett. .

Abstract

The ability of chemo-radiation therapy to control locally advanced stage III non-small cell lung cancer (NSCLC) is poor. While addition of consolidation immunotherapy has improved outcomes in subsets of patients there is still an urgent need for new therapeutic targets. Emerging research indicates that nucleophosmin1 (NPM1) is over-expressed in NSCLC, promotes tumor growth and that over-expression correlates with a lower survival probability. NPM1 is critical for APE1 base excision activity and for RAD51-mediated repair of DNA double strand breaks (DSBs). YTR107 is a small molecule radiation sensitizer that has been shown to bind to NPM1, suppressing pentamer formation. Here we show that in irradiated cells YTR107 inhibits SUMOylated NPM1 from associating with RAD51, RAD51 foci formation and repair of DSBs. YTR107 acts synergistically with the PARP1/2 inhibitor ABT 888 to increase replication stress and radiation-induced cell lethality. YTR107 was found to radiosensitize tumor initiating cells. Congruent with this knowledge, adding YTR107 to a fractionated irradiation regimen diminished NSCLC xenograft growth and increased overall survival. These data support the hypothesis that YTR107 represents a therapeutic target for control of NSCLC.

Keywords: NSCLC; Nucleophosmin1; RAD51; Radiation sensitization; YTR107.

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Conflict of interest statement

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1.
Fig. 1.
YTR107 inhibits NPM1/RAD51 interactions. A) HEK293 cells were transfected with a plasmid that expressed a chimeric GFP/FLAG/NPM1 protein. Five days after transfection the cells were exposed to 0 or 35 μM YTR107 for 30 min prior to administering 0 or 10 Gy. Two hrs later cells were solubilized in 0.5% NP40 buffer. Protein was immunoprecipitated using a RAD51 antibody. Immunoprecipitated protein was immunoblotted with FLAG antibody. The expression of immunoprecipitated GFP/FLAG/NPM1 relative to immunoprecipitated RAD51 is shown at the top of figure. B) HEK293 cells were transfected with a plasmid that expressed a chimeric GFP/FLAG/NPM1 protein. Five days after transfection the cells were exposed to 0 or 25 μM YTR107 for 30 min prior to administering 0 or 10 Gy. Two hrs later cells were solubilized in 0.5% NP40 buffer. Protein was immunoprecipitated using a SUMO-1 antibody. Immunoprecipitated protein was immunoblotted with FLAG antibody.
Fig. 2.
Fig. 2.
RAD51 foci formation is inhibited by YTR107. (A) A549 cells were exposed to 25 μM YTR107 for 30 min prior to, during and for 4 h after 0 or 10 Gy. Cells were immunostained for RAD51 and DAPI counterstained. (B) Quantification of individual foci per nuclei is expressed as box and whisker plots, max to min, and represents the average of four independent experiments. N = 132 for 0 Gy DMSO; N = 109 for 0 Gy YTR107; N = 365 for 10 Gy (DMSO); N = 208 for 10 Gy YTR107. (C) Cells were immunostained for RAD 51 and Geminin, a S/G2 phase marker. (D) Quantification of individual RAD51 foci per nuclei in Geminin expressing cells. white line = 10 μm.
Fig. 3.
Fig. 3.
YTR107 inhibits repair of DSBs. H460 cells were exposed to 25 μM YTR107 for 30 min prior to, during administration of 4 Gy and for 45 min after γ-irradiation. Cells were then subjected to neutral comet assays. (A) Quantification of mean tail moment. Red bar = mean. (B) Representative comet images.
Fig. 4.
Fig. 4.
YTR107-mediated radiosensitization. Calu1 (A), A549 (B), and H460 (C) cells were exposed to YTR107 for 30 min prior to, during and or only 90 min after irradiation. Clonal survival to YTR107 alone was: 38%, 72% and 59% for Calu1, A549, and H460 respectively. Colony formation assays were used to generate the survival curves fitted by least squares to equation S = e−αD−βD^2. The dose response curves shown in the circles illustrates survival as a function of dose up to 4 Gy. (D–F) For tumorsphere assays A549, H226, and H1975 cells were exposed to YTR107 (35 μM) or DMSO for 30 min prior to, during and for 90 min after irradiation (5 Gy). After extensive washing, varying cell numbers were inoculated into ultra-low adhesion wells for tumorsphere limiting dilution assays. (G & H) Monolayers (5 × 106 cells) of A549 were exposed to DMSO, the solvent control, or YTR107 (35 μM) for 30 min before, during and for 90 min after 0 or 5 Gy and then washed extensively. Then 500 washed cells (G) or varying numbers of washed cells (H) were injected into the flanks of athymic nude mice. Tumor formation was monitored for 40 days. TIC – Tumor Initiating Cells, P value compares 1/TIC Frequency for 5 Gy alone + DMSO vs 5 Gy + YTR107.
Fig. 5.
Fig. 5.
NSCLC xenograft growth delay is enhanced by YTR107. A549 xenograft-bearing mice were subjected to a 7-day regimen consisting of YTR107 (20 mg/kg) or solvent control (DMSO) administered IP 1 h before tumors received 0 or 2.2-Gy. Tumor growth curves are shown in panel (A). Days (±SD) required to achieve a 5-fold increase tumor volume are plotted in (B). (C) Kaplan Meier survival analysis. A549 xenograft-bearing mice were subjected to a 7-day regimen consisting of YTR107 (20 mg/kg) or solvent control (DMSO) administered IP 1 h before tumors received 0 or 2.2-Gy.
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References

    1. Ettinger DS, Aisner DL, Wood DE, Akerley W, Bauman J, Chang JY, Chirieac LR, D’Amico TA, Dilling TJ, Dobelbower M, Govindan R, Gubens MA, Hennon M, Horn L, Lackner RP, Lanuti M, Leal TA, Lilenbaum R, Lin J, Loo BW, Martins R, Otterson GA, Patel SP, Reckamp K, Riely GJ, Schild SE, Shapiro TA, Stevenson J, Swanson SJ, Tauer K, Yang SC, Gregory K, Hughes M, NCCN guidelines insights: non-small cell lung cancer, version 5.2018, J. Natl. Compr. Canc. Netw 16 (2018) 807–821. - PubMed
    1. Sharma RA, Plummer R, Stock JK, Greenhalgh TA, Ataman O, Kelly S, Clay R, Adams RA, Baird RD, Billingham L, Brown SR, Buckland S, Bulbeck H, Chalmers AJ, Clack G, Cranston AN, Damstrup L, Ferraldeschi R, Forster MD, Golec J, Hagan RM, Hall E, Hanauske AR, Harrington KJ, Haswell T, Hawkins MA, Illidge T, Jones H, Kennedy AS, McDonald F, Melcher T, O’Connor JP, Pollard JR, Saunders MP, Sebag-Montefiore D, Smitt M, Staffurth J, Stratford IJ, Wedge SR, Group NCA-PJW, Clinical development of new drug-radiotherapy combinations, Nat. Rev. Clin. Oncol 13 (2016) 627–642. - PubMed
    1. Walker AJ, Kim H, Saber H, Kluetz PG, Kim G, Pazdur R, Clinical development of cancer drugs in combination with external beam radiation therapy: US food and drug administration perspective, Int. J. Radiat. Oncol. Biol. Phys 98 (2017) 5–7. - PubMed
    1. Jabbour SK, Berman AT, Simone CB 2nd, Integrating immunotherapy into chemoradiation regimens for medically inoperable locally advanced non-small cell lung cancer, Transl. Lung Cancer Res 6 (2017) 113–118. - PMC - PubMed
    1. Gray JE, Villegas A, Daniel D, Vicente D, Murakami S, Hui R, Kurata T, Chiappori A, Lee KH, Cho BC, Planchard D, Paz-Ares L, Faivre-Finn C, Vansteenkiste JF, Spigel DR, Wadsworth C, Taboada M, Dennis PA, Ozguroglu M, Antonia SJ, Three-year overall survival with durvalumab after chemoradiotherapy in stage III NSCLC-update from PACIFIC, J. Thorac. Oncol 15 (2020) 288–293. - PMC - PubMed

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