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An IRAK1–PIN1 signalling axis drives intrinsic tumour resistance to radiation therapy

Nature Cell Biology volume 21, pages 203–213 (2019)Cite this article

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Abstract

Drug-based strategies to overcome tumour resistance to radiotherapy (R-RT) remain limited by the single-agent toxicity of traditional radiosensitizers (for example, platinums) and a lack of targeted alternatives. In a screen for compounds that restore radiosensitivity in p53 mutant zebrafish while tolerated in non-irradiated wild-type animals, we identified the benzimidazole anthelmintic oxfendazole. Surprisingly, oxfendazole acts via the inhibition of IRAK1, a kinase thus far implicated in interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) immune responses. IRAK1 drives R-RT in a pathway involving IRAK4 and TRAF6 but not the IL-1R/TLR–IRAK adaptor MyD88. Rather than stimulating nuclear factor-κB, radiation-activated IRAK1 prevented apoptosis mediated by the PIDDosome complex (comprising PIDD, RAIDD and caspase-2). Countering this pathway with IRAK1 inhibitors suppressed R-RT in tumour models derived from cancers in which TP53 mutations predict R-RT. Moreover, IRAK1 inhibitors synergized with inhibitors of PIN1, a prolyl isomerase essential for IRAK1 activation in response to pathogens and, as shown here, in response to ionizing radiation. These data identify an IRAK1 radiation-response pathway as a rational chemoradiation therapy target.

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Fig. 1: In vivo zebrafish drug screen identifies oxfendazole as a radiosensitizer of p53 mutant embryos.
Fig. 2: Target discovery for oxfendazole identifies IRAK1.
Fig. 3: Targeting irak1 overcomes R-RT in p53MK/MK zebrafish.
Fig. 4: IRAK1 acts independently of MyD88 and counters PIDDosome signalling.
Fig. 5: IRAK1 inhibitors restore radiosensitivity across TP53 mutant tumour cell models.
Fig. 6: PIN1 inhibition overcomes R-RT in zebrafish and human tumour-cell models while its overexpression associates with TP53 mutant HNSCC recurrence.
Fig. 7: Low-dose IRAK1 and PIN1 inhibitors synergistically suppress R-RT in vitro and in vivo.

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Code availability

All codes used are referenced in the Methods sections above.

Data availability

The whole-genome expression array data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE67614. The human HNSCC and breast cancer data were derived from TCGA Research Network: http://cancergenome.nih.gov/. The dataset derived from this resource that supports the findings of this study is available in Supplementary Table 4 (see sheet S3). Source data for Figs. 1e, 2b,d, 3c,e,f, 4a–e,g,h,m,n, 5b,d, 6c–j, 7a–d,f,g and Supplementary Figs. 1b, 2a,b, 3, 4a–f,k,l, 5, 6c–n and 7c,d are provided as Supplementary Table 4. The full image dataset that supports the findings in Fig. 3e,f has been deposited in Figshare at https://doi.org/10.6084/m9.figshare.7427942. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Change history

  • 01 February 2019

    In the sentence beginning “Moreover, IRAK1 inhibitors...” in the Abstract of the version of this Article originally published, it was unclear whether the present study shows synergy between IRAK1 and PIN1 inhibitors in response to pathogens or ionizing radiation. The sentence has been modified to clarify this.

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Acknowledgements

The authors thank E. Farias, R. Cagan, A. Dar, R. Krauss, J. Ashwell, X. Li, M. Posner, P. Reddy, R. De Vita and R. Sanchez for helpful comments, technical advice and reagents, and C. Franco and D. Dominguez for zebrafish care. This work was supported in part by the following bodies: NIH/NCI (F30CA186448 to P.H.L.; P30 CA008748 to K.S.P.; RO1CA168485 to H.D.S. and J.V.H; and RO1CA178162 to S.S.); the National Cancer Center NCC CCP (postdoctoral fellowship (to Y.L.); and the JJR Foundation, the Pershing Square Sohn Cancer Research Alliance, the New York Community Trust and the Searle Scholars Program (to S.S.).

Author information

Author notes

  1. Vincent Brechin

    Present address: Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan

  2. John M. Barbaro

    Present address: Albert Einstein College of Medicine, Bronx, NY, USA

  3. Ruth Thompson

    Present address: Department of Oncology and Metabolism, The University of Sheffield, Sheffield, UK

  4. Robert G. Maki

    Present address: Hofstra-Northwell School of Medicine and Cold Spring Harbor Laboratory, Hempstead, NY, USA

Authors and Affiliations

  1. Department of Medicine, Division of Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Peter H. Liu, Richa B. Shah, Yuanyuan Li, Renuka Raman, Vincent Brechin, John M. Barbaro, Ruth Thompson, Julio A. Aguirre-Ghiso, Robert G. Maki & Samuel Sidi

  2. Department of Cell, Developmental and Regenerative Biology, The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Peter H. Liu, Richa B. Shah, Yuanyuan Li, Renuka Raman, Vincent Brechin, John M. Barbaro, Ruth Thompson, Robert G. Maki & Samuel Sidi

  3. Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Arshi Arora & Katherine S. Panageas

  4. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Peter Man-Un Ung & Avner Schlessinger

  5. Department of Pathology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Andrej Gorbatenko & Jose M. Silva

  6. Cancer Biology Program, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Shingo Kozono, Xiao Zhen Zhou & Kun Ping Lu

  7. Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Richard M. White

  8. Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    John V. Heymach & Heath D. Skinner

  9. Antitumor Assessment Core and Molecular Pharmacology Department, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Elisa de Stanchina

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Contributions

P.H.L. performed the following experiments: zebrafish drug screen with the assistance of J.M.B and V.B.; all zebrafish experiments with the exception of the TUNEL and caspase-3 assays (V.B.), and the p53atgMO, myd88MO, and hIRAK1 WT, K/R and E3A rescue assays (Y.L); and SEA analyses under the supervision of A.S. and S.S. R.B.S. performed all the cell culture assays with the assistance of P.H.L. and R.T., and analysed the data with P.H.L., Y.L. and S.S. R.G.M. purchased the radioresistant cell lines and assisted with translational analyses with J.A.A.-G. E.d.S., R.M.W., J.A.A.-G. and S.S. conceived the xenograft study, which was performed by E.d.S. and analysed by R.R. A.A. performed TCGA analyses under the supervision of K.S.P. and analysed the data with R.M.W. and S.S. P.M.-U.U. performed docking studies under the supervision of A.S. A.G. generated the IRAK1 KO under the supervision of J.M.S. S.K. and X.Z.Z. performed the in vitro isomerase assays under the supervision of K.P.L. H.D.S. analysed PIN1 expression levels in the MD Anderson Cancer Center HNSCC cohort with the assistance of J.V.H. S.S. conceived the study, supervised the project and wrote the paper.

Corresponding author

Correspondence to Samuel Sidi.

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Integrated supplementary information

Supplementary Figure 1 Extended data for Main Fig. 1.

(a) Representative images of 25 hpf embryos stained with AO, with conditions indicated, 7 hpIR (15 Gy Cs γ-IR TBI delivered at 18 hpf). Scale bar, 0.5 mm. (b) Quantification of AO positivity in the neural tubes of embryos from panel (a). Number of quantified embryos were: p53+/+, DMSO-treated: n of non-irradiated = 7, n of irradiated = 7; p53+/+, Gö6976-treated: n of non-irradiated = 6, n of irradiated = 8. p53MK/MK, DMSO-treated: n of non-irradiated = 7, n of irradiated = 8; p53MK/MK, Gö6976-treated: n of non-irradiated = 7, n of irradiated = 8; altogether obtained from 2 independent experiments. Data expressed as means ± SD, ***P < 0.0001, n.s., not significant, two-tailed Student’s t-test. See Supplementary Table 4 for statistics source data including precise P values. (c) Schematic of primary screen. Embryos arrayed in multi-well plates and treated with drug libraries at 17 hpf, irradiated (15 Gy) at 18 hpf, drug-treated for 6 hr, washout at 24 hpf, and scored for DTCs at 120 hpf. (d) Primary screen results. 640 FDA-approved drugs (X-axis, 2 μg/mL each) scored for DTC penetrance (Y-axis) in >12 embryos/drug. Gö6976, used as positive control, labeled in red. Drugs producing >75% DTC were considered primary hits and carried over to secondary screen. (e) Secondary screen. For each primary hit in (d), embryos were arrayed in a similar schema but half of embryos were non-irradiated. Results plotted in (f-g) below. (f) Schematic of 136 drugs screened in secondary screen (e). Quadrants determined based on greater than or less than 50% potency (%DTC +IR) and greater or less than a selectivity ratio [(%DTC +IR)/(%DTC –IR)] of 4:1. Quadrant I: selective but not potent; II: both potent and selective; III: potent but not selective; IV: neither potent nor selective. Identity of each compound in the screen described in Supplementary Tables 1-2. (g) Each drug was investigated by SEA for possibly targeting IRAK1/4 or PIN1. Red, predicted to bind IRAK1/4; blue, predicted to bind PIN1/4; purple, predicted to bind both IRAK1/4 and PIN1/4. Additional, possibly lethal off-targets were not investigated.

Supplementary Figure 2 Extended data for Main Figs. 1 and 2.

(a) Representative images of anterior spinal cords (as depicted on top) of WT and p53MK/MK embryos injected with standard control MO (std MO) or p53 MO targeted to initiator ATG (p53 MO (atg)) and treated with 15 Gy IR with or without oxfendazole (20 µg/ml) at 18 hpf. Embryos stained with AO at 7.5 hpIR. (b) Quantification of spinal cord areas shown in (a). Data represented as means ± SD of n = 3 independent experiments with at least 8 spinal chord images analyzed per condition; ***, P < 0.0005, ns, non-significant, two-tailed student’s t test. See Supplementary Table 4 for statistics source data including precise P values. (c-d) Pooled embryonic lysates analyzed by western blot with indicated antibodies. (e) Confocal microscopy of p53MK/MK zebrafish tails stained with DAPI (blue) and anti-γH2AX (green) after 0 Gy or 15 Gy IR and DMSO vs oxfendazole (20 µg/ml). Arrowheads indicates positively staining cells. hpIR, hours post ionizing radiation. hpf, hours post fertilization. (f) HeLa cells treated with indicated IRAK1 inhibitors or PIN1 inhibitor (EGCG) were irradiated (10 Gy), harvested at indicated time points and analyzed by western blot with indicated antibodies. (g) Acridine orange (AO) staining of 48 hpf embryos after 0 or 15 Gy IR and increasing doses of oxfendazole, with magnified views of spinal cords shown below. (h) Magnified images of spinal cords stained with AO at 6 hpIR, 18 hpIR, and 30 hpIR. (i) Oxfendazole (20 µg/ml) suppression of R-RT requires a 4-hr post-IR window as shown through a temporal requirement experiment. Readouts of R-RT observed as penetrance (%, absolute numbers) after AO staining at 48 hpf and DTC at 120 hpf with drug incubation windows depicted in green. (j) Quantification of DTCs in >12 p53MK/MK embryos at 120 hpf treated with inhibitors to SEA-predicted oxfendazole targets and irradiated at 18 hpf. Highest doses tested here tested in Fig. 2b. Shown are means of n = 2 independent experiments. Scale bars, 0.2 (a,g-h) and 0.1 (e) mm. See Supplementary Fig. 8 for unprocessed immunoblots.

Supplementary Figure 3 IRAK1 and PIN1 mRNA expression levels in HNSCC and BC TCGA cohorts.

The Cancer Genome Atlas (TCGA) analysis shows the relative mRNA levels of IRAK1 (a-d, i-l) and PIN1 (e-h, m-p) in HNSCC (a-h) and BC (i-p) as a function of TP53 status (a, e, i, m), future RT treatment or lack thereof (b, f, j, n), or both TP53 status and future RT data sets (c-d, g-h, k-l, o-p). The n number of independent patient samples is indicated in each panel. *P < 0.05, **P < 0.005, ***P < 0.0001, NS, not significant; two sided Wilcoxon rank-sum test. See Supplementary Table 4 for statistics source data including precise P values.

Supplementary Figure 4 Extended data for main Figs. 4 and 5.

(a-b) AlamarBlue survival curves of SAS, MDA-MB-231 and CAL27 shTRIPZ (control) and shIRAK1 cells (a) and IRAK1/4i-treated HeLa cells (b) at indicated IR doses. Western blots showing shRNA knockdown efficacy also shown in (a). All data represented as means ± SD, n = 3 independent experiments performed in triplicate, *P < 0.05, **P < 0.005, ***P < 0.0005, two-tailed Student’s t-test. (c-f) Clonogenic assays of HeLa (c-d), CAL27 (e) and SAS (f) cells either stably expressing IRAK1 shRNA (c) or treated with R406 (1 μg/ml, d-f) and irradiated with indicated γIR doses (Gy). Representative images shown to the left, surviving fractions to the right. All data represented as means ± SD, n = 3 independent experiments performed in triplicate, *P < 0.05, **P < 0.005, ***P < 0.0005, two-tailed Student’s t-test. (g) HeLa cells transfected with control LACZ or MyD88 siRNA and treated with IL-1β (100 ng/ml) analyzed by immunofluorescence with indicated antibodies 5 minutes after stimulation. siMyD88 abrogates recruitment of IRAK1 to cell surface, and is thus sufficient to eliminate MyD88 function. Scale bar, 20 μm. (h) Control and IRAK1 shRNA HeLa cells treated with IL-1β (100 ng/ml) or IR (10 Gy), harvested at indicated time points after stimulus and analyzed by western blot. (i) Extended data for western blot shown in Fig. 4k to show RAIDD, Chk2 phosphorylation (marker of ATM activity), total Chk2 and GAPDH levels. (j) Extended data for western blot shown in Fig. 4d to show caspase-2 cleavage levels. (k-l) Quantification and representative images of p53MK/MK embryos injected with indicated MOs, irradiated (15 Gy) at 18 hpf and scored for DTCs at 5 dpf. Data in (k) are means ± SD of n = 3 independent experiments (bars 3,4,7,8) or n = 4 independent experiments (bars 1,2,5,6). *P < 0.05; ***P < 0.0005; ns, not significant, two-tailed Student’s t-test. Scale bar, 0.5 mm. See Supplementary Table 4 and Supplementary Figure 8 for statistics source data including precise P values and unprocessed immunoblots, respectively.

Supplementary Figure 5 Extended data for main Fig. 5.

Line graph representations of alamarBlue-based cell viability assays shown in Fig. 5d-f. Indicated cancer-cell lines after treatment with cisplatin, ginsenoside Rb1, and R406 after 0, 2.5, 5, and 7.5 Gy IR. TP53 status indicated next to cell line name. Data represented as means of n = 3 independent experiments, *P < 0.05; **P < 0.005; ***P < 0.0005; ns, not significant, two-tailed Student’s t-test. See Supplementary Table 4 for statistics source data including precise P values.

Supplementary Figure 6 Extended data for main Fig. 6.

(a) PPIase, cyclophilin and FKBP12 enzymatic assays of oxfendazole (top row) and corresponding positive controls (bottom row). Fluorescein absorbance measured every 3 seconds. (b) Thermal shift assay. PIN1 alone (blue) or with indicated drugs in two replicates. Tm, melting temperature. (c) Quantification of DTCs represented in Fig. 6a; n = 4 independent experiments (bars 2 and 4) and n = 3 independent experiments (bars 1,2,5–8). (d) Quantification of individual embryos as shown in Fig. 6b. Number of quantified embryo images over 2 experiments: DMSO-treated: n of non-irradiated = 2, n of irradiated = 5; oxfendazole-treated: n of non-irradiated = 4, n of irradiated = 5; stdMO-treated: n of non-irradiated = 5, n of irradiated = 7; pin1MO-treated: n of non-irradiated = 7, n of irradiated = 7. (e) PIN1 MO dose-response curve as scored in DTC assay; n = 4 independent experiments (0.0 mM), n = 3 independent experiments (0.2 mM) and n = 2 independent experiments (0.1, 0.3 and 0.4 mM). (f-g) Dose-response DTC curves for indicated PIN1 inhibitors, >12 embryos scored per condition; n = 3 independent experiments. (h) Oxfendazole synergizes with EGCG at doses which independently do not produce DTCs. Data are means of 2 independent experiments. (i) TUNEL assay of HeLa cells after 10 Gy IR with indicated siRNA or oxfendazole; n = 3 independent experiments in triplicates. (j) Clonogenic assay of HeLa cells treated with buparvaqone (5 μg/ml) and up to 5 Gy IR; n = 5 independent experiments in triplicates. (k) shTRIPZ (control) and shPIN1 CAL27 cells treated with indicated IR doses assayed by alamarBlue at 5 dpIR; n = 3 independent experiments in triplicates. Western blot showing PIN1 knockdown also shown. (l - m) Clonogenic assays of CAL27 (l) and SAS cells (m) treated with buparvaquone (5 μg/ml); n = 5 independent experiments in triplicates. (n) Survival curves for Fig. 6f; n = 3 independent experiments in triplicates. All data represented as means ± SD, *P < 0.05, **P < 0.005, ***P < 0.0005, two-tailed Student’s t-test. See Supplementary Table 4 for statistics source data including precise P values.

Supplementary Figure 7 Extended data for Main Fig. 7.

(a) HeLa cells treated as in Fig. 7a analyzed by alamarBlue at 5 dpIR. Data expressed as means of 2 independent experiments in triplicates. CI, combination index, see (e). (b) Indicated cell lines treated and analyzed as in (a) with buparvaquone (0.5 µg/mL in BHY, 1 µg/mL in CAL27, SAS, and SW480) and R406 (0.1 µg/mL in BHY, 0.5 µg/mL in CAL27, SAS, and SW480). Data expressed as means of 2 independent experiments in triplicates. CI, see (g). (c-d) Clonogenic assays of CAL27 (c) and SAS (d) cells treated with subtherapeutic concentrations of the indicated drugs alone or in combination with IR (Gy); buparvaquone: 0.5 µg/mL (BHY), 1 µg/mL otherwise; R406: 0.1 µg/mL (BHY), 0.5 µg/mL otherwise. Data represented as means ± SD of n = 3 independent experiments in triplicates. (e-g) CI values for panel a (e), Fig. 7c (f) and panel b (g). (e) Each drug tested at two doses, thereby representing four data points after treatment with 0, 2.5, or 5 Gy IR. After 2.5 or 5 Gy IR, every combination of IRAK1+PIN1 inhibitor produced CI’s <0.8–1, indicating synergy. (f) CI values for p53MK/MK embryos treated with four possible combinations of two doses of EGCG and R406. At greater than 50% fractions affected, EGCG + R406 combination showed <0.8 CI, indicating synergy. Bottom-right data point is dose combination in Fig. 7c. (g) CI values for BHY, CAL27, SAS, and SW480 cells after indicated drug/IR combinations. Each data point represents mean of n = 3 independent experiments. (h) Extended data for Fig. 7e-g showing tumor volume (top) and animal weight (bottom) assays. Data expressed as means ± SEM, n = 5 mice per group. (i) Cells treated and analyzed as in (a), but substituting TP53 WT and null HCT116 cells for HeLa. Subtherapeutic concentrations as established for HCT116 cells were: R406, 0.1 μg/ml; Gin-Rb1, 5 μg/ml; buparvaquone 0.5 μg/ml; EGCG, 5 μg/ml. Data expressed as means of 2 independent experiments in triplicates. *P <0.05, **P <0.005, ***P <0.0005, two-tailed Student’s t-test. See Supplementary Table 4 for statistics source data including precise P values.

Supplementary Figure 8 Unprocessed immunoblots.

Unprocessed immunoblots for indicated Figures panels.

Supplementary Figure 9 Unprocessed immunoblots.

Unprocessed immunoblots for indicated Figures panels.

Supplementary Figure 10 Unprocessed immunoblots.

Unprocessed immunoblots for indicated Figures panels.

Supplementary information

Supplementary Information

Supplementary Figures 1–8 and Supplementary Table legends.

Reporting Summary

Supplementary Table 1

Contents of FDA-approved library with secondary screen quadrant status.

Supplementary Table 2

Small molecule screen information.

Supplementary Table 3

Patient tumour characteristics.

Supplementary Table 4

Statistics source data.

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Liu, P.H., Shah, R.B., Li, Y. et al. An IRAK1–PIN1 signalling axis drives intrinsic tumour resistance to radiation therapy. Nat Cell Biol 21, 203–213 (2019). https://doi.org/10.1038/s41556-018-0260-7

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