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. 2010 Aug 1;70(15):6268-76.
doi: 10.1158/0008-5472.CAN-09-3416. Epub 2010 Jul 14.

6-thioguanine selectively kills BRCA2-defective tumors and overcomes PARP inhibitor resistance

Natalia Issaeva  1 , Huw D Thomas, Tatjana Djureinovic, Janneke E Jaspers, Ivaylo Stoimenov, Suzanne Kyle, Nicholas Pedley, Ponnari Gottipati, Rafal Zur, Kate Sleeth, Vicky Chatzakos, Evan A Mulligan, Cecilia Lundin, Evgenia Gubanova, Ariena Kersbergen, Adrian L Harris, Ricky A Sharma, Sven Rottenberg, Nicola J Curtin, Thomas Helleday
Affiliations

6-thioguanine selectively kills BRCA2-defective tumors and overcomes PARP inhibitor resistance

Natalia Issaeva et al. Cancer Res. .

Erratum in

  • Cancer Res. 2010 Oct 1;70(19):7734. Djurenovic, Tatjana [corrected to Djureinovic, Tatjana]

Abstract

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.

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Figures

Figure 1
Figure 1. homologous recombination defective cells are hypersensitive to chemical 6TG
(a) 6TG selectively kill BRCA2 defective VC-8 cells in a colony formation assay. 6TG-induces apoptosis in HR defective VC-8 cells as measured by FACS analysis of the (b) subG1 population and (c) TUNEL staining. The average and standard deviation from at least three experiments is shown. (d) Tumour outgrowth in xenograft mice (5 per group) following injection of V-C8 and BRCA2 complemented V-C8+B2 upon i.p. treatment with 6TG and PARP inhibitor.
Figure 2
Figure 2. homologous recombination is needed to repair the damage induced by 6TG
Equal amount of DNA damage in V-C8 and V-C8+B2 cells following 6-thioguanine treatment, as determined by (a) γH2AX foci (Cells containing more than 10 foci were scored as positive) or (b) by Western blot. (c) RAD51 foci do not form in V-C8 cells upon 6TG induced DNA damage. RAD51 foci do form in V-C8+B2 cells and co-localise with γH2AX foci. (d) Quantification of RAD51 foci formed in V-C8 and V-C8+B2 cells after 6TG treatment. (e) 6TG selectively kills XRCC3 defective irs1SF cells in a colony formation assay as compared to wild type control (AA8).
Figure 3
Figure 3. Response of the PARP inhibitor resistant Brca1Δ5-13/Δ5-13;p53Δ2-10/Δ2-10 tumour T6-28 to 6TG
Animals carrying orthotopically transplanted tumours were treated with 1.5mg 6TG per kg i.p. daily on days 0-9 or 50mg olaparib per kg i.p. daily when the tumours reached a volume of 150-250mm3 (100% day0). When tumours relapsed back to 100% (arrows), a second treatment of 1.5mg 6TG per kg daily for 4 consecutive days was tolerated. rs.
Figure 4
Figure 4. PARP-inhibitor and cisplatin- resistant BRCA2 defective cells and tumours respond to 6-thioguanine
Clonogenic survival in BRCA2 defective V-C8, BRCA2 complemented V-C8+B2 and V-C8 PARP inhibitor resistant clones following treatment (a) PARP inhibitor AG014699; (b) cisplatin; (c) 6TG. The average and standard deviation of at least three experiments is shown. (d) Tumour outgrowth in xenograft mice following injection of PIR V-C8 clone 2B upon i.p. treatment with 6TG and PARP inhibitor. 6TG retards PIR V-C8 clone 2B tumour outgrowth (statistically significant in Mann Whitney test p<0.01). The average and standard error from ten mice in each group is shown.
Figure 5
Figure 5. PARP-inhibitor resistant BRCA2 defective cells partially activates HR in response to 6TG
(a) Quantification of 6TG and PARP inhibitor induced RAD51 foci. BRCA2 complemented V-C8+B2 cells showed a significant increase in RAD51 foci compared to PIR V-C8 cells upon 6TG treatment (24 hours after an 18 hour treatment with 1μM of 6-TG) (b) Quantification of repair of 6TG-induced γH2AX foci. HR defective V-C8 cells fail to repair 6TG-induced γH2AX foci, while V-C8+B2 cells repairs a majority of 6TG-induced γH2AX foci. PIR V-C8 clones show an intermediate repair of 6TG-induced γH2AX foci. The average and standard error of three independent experiments is depicted. Values marked with asterisks are statistically significant in T-test (* p<0.05; ** p<0.01; *** p<0.001).
Figure 6
Figure 6. BRCA2 suppresses 6TG toxicity in mismatch repair deficient cells
(a) Survival following continuous treatment with PARP inhibitor ANI, 6TG and cisplatin in HCT116 and HCT116+Ch3 cells. (b) γH2AX foci formation in hMLH1 defective HCT116 and hMLH1 complemented HCT116+Ch3 cells after a 24 hour treatment with 6TG and cisplatin or a 4 hour treatment with PARP inhibitor ANI. (c) Clonogenic survival in HCT116 and BRCA2 depleted HCT116 to increasing doses of 6TG. The average and standard deviation of three independent experiments is depicted. Values marked with asterisks are statistically significant in T-test (*** p<0.001).
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References

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