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. 2015 Apr 15;21(8):1962-72.
doi: 10.1158/1078-0432.CCR-14-2616. Epub 2015 Jan 21.

Acquisition of Relative Interstrand Crosslinker Resistance and PARP Inhibitor Sensitivity in Fanconi Anemia Head and Neck Cancers

Anne J Lombardi  1 , Elizabeth E Hoskins  1 , Grant D Foglesong  1 , Kathryn A Wikenheiser-Brokamp  2 , Lisa Wiesmüller  3 , Helmut Hanenberg  4 , Paul R Andreassen  1 , Allison J Jacobs  5 , Susan B Olson  6 , Winifred W Keeble  5 , Laura E Hays  7 , Susanne I Wells  8
Affiliations

Acquisition of Relative Interstrand Crosslinker Resistance and PARP Inhibitor Sensitivity in Fanconi Anemia Head and Neck Cancers

Anne J Lombardi et al. Clin Cancer Res. .

Abstract

Purpose: Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease.

Experimental design: Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc(-/-) mice, we sought to define Fanconi anemia-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ).

Results: Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia-deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation.

Conclusions: The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual.

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Figures

Figure 1
Figure 1. Gene correction of FA patient-derived HNSCC cell lines
A) Immunoblot of isogenic FA patient-derived HNSCC cell lines. Complementation of the relevant FA gene restores FANCD2 activity upon treatment with hydroxyurea (HU). FAmut: FA-deficient; FAcomp: FA-complemented; S: FANCD2; L: Monoubiquitinated FANCD2. B) Immunofluorescence for FANCD2 and γH2AX shows localization of FANCD2 to sites of DNA damage in FAcomp cells following MMC treatment. Images shown are representative of three independent experiments, each with similar results. C) FAcomp cells are rescued from cell cycle arrest in the G2/M phase caused by melphalan treatment.
Figure 2
Figure 2. Three-dimensional organotypic epithelial rafts generated from human FA HNSCC cells
A) H&E and immunofluorescence staining of rafts created from isogenic HNSCC cell lines. Images are representative of three independent experiments, each with similar results. H&E sections of FAmut and FAcomp rafts are equivalent in mitotic index, cellular differentiation, and stromal content. Immunofluorescence for BrdU incorporation indicates similar proliferative rates. B) Quantification of BrdU incorporation of FAmut and FAcomp cells; a t-test indicated no significant difference.
Figure 3
Figure 3. Interstrand crosslinker sensitivity of murine FA HNSCC
A) Cisplatin (left) and MMC (right) cellular growth assays of immortalized, non-malignant oral epithelial cells of Fancc−/− (M-NR) and WT (W-NR) mice shows significantly increased sensitivity of Fancc−/− cell lines. B) MMC half-maximal effective concentrations (EC50s) of immortalized, non-malignant murine Fancc−/− and WT oral epithelial cells. ** p < 0.01 (t-test). C) Cisplatin (left) and MMC (right) cellular growth assays of murine Fancc−/− (M-SCC) and WT (WSCC) mice indicate overlap of sensitivities. D) MMC EC50s of Fancc−/− and WT HNSCC cell lines following 5 days of exposure; a t-test revealed no significant difference.
Figure 4
Figure 4. Interstrand crosslinker sensitivity of human FA HNSCC
A) MMC cellular growth assays of FA patient-derived (black) and sporadic (gray) HNSCC cell lines indicate overlap of sensitivities following 5 days of treatment. B) MMC EC50s of FA patient-derived and sporadic HNSCC cell lines following 5 days of exposure; a t-test revealed no significant difference.
Figure 5
Figure 5. Preference for NHEJ of mutant and complemented FA HNSCC cells
A) Schematic of DNA repair reporter assay constructs for NHEJ. B) Reporter assays performed on isogenic VU-1131 cells indicate that FA gene correction decreases baseline preference for NHEJ. ** p < 0.01 (t-test). C) Immunoblot of total DNA-PKcs and pDNA-PKcsS2056 in FAmut and FAcomp cell lines treated with 2 μg/mL bleomycin for 20 minutes in the presence and absence of 24 hours pre-treatment with the DNA-PKcs inhibitor NU-7026 (2 μM). Pretreatment with NU-7026 decreases phosphorylation of DNA-PKcs caused by bleomycin treatment. D) Chemical inhibition of DNA-PKcs does not decrease the cisplatin EC50 of FAmut HNSCC cells relative to FAcomp cells following 2 days of exposure.
Figure 6
Figure 6. PARP inhibitor sensitivity of human and murine FA HNSCC cells
A) Cellular growth assays on isogenic human HNSCC cells exposed to the PARP1/PARP2 inhibitor olaparib shows uniform sensitivity of FAmut cell lines. * p < 0.05; ** p < 0.01 (t-test). B) Immunofluorescence for PAR foci in VU-1131 cells shows increased PAR foci formation in FAmut cells over a 24-hour course of MMC treatment. Results shown are representative of two (no treatment) or three (2, 4, and 24 hours) independent experiments of each time point, each with similar results. C) Quantification of intranuclear PAR foci in VU-1131 cells over a 24-hour course of MMC treatment reveals a significantly increased number of PAR foci in FAmut cells at 4 hours of exposure. ** p < 0.01 (t-test). D) Cellular growth assays performed on murine Fancc−/− (M-SCC) and WT (W-SCC) HNSCC cell lines treated with olaparib. shows significantly increased sensitivity of Fancc−/− cell lines. * p < 0.05.
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