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. 2020 Feb 19;12(531):eaax2625.
doi: 10.1126/scitranslmed.aax2625.

BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers

Florence Coussy  1   2   3 , Rania El-Botty  1 , Sophie Château-Joubert  4 , Ahmed Dahmani  1 , Elodie Montaudon  1 , Sophie Leboucher  5 , Ludivine Morisset  1 , Pierre Painsec  1 , Laura Sourd  1 , Léa Huguet  1 , Fariba Nemati  1 , Jean-Luc Servely  4   6 , Thibaut Larcher  7 , Sophie Vacher  3 , Adrien Briaux  3 , Cécile Reyes  1 , Philippe La Rosa  8   9 , Georges Lucotte  8   9 , Tatiana Popova  9   10 , Pierre Foidart  11 , Nor Eddine Sounni  11 , Agnès Noel  11 , Didier Decaudin  1   2 , Laetitia Fuhrmann  12 , Anne Salomon  12 , Fabien Reyal  13   14 , Christopher Mueller  15 , Petra Ter Brugge  16 , Jos Jonkers  16 , Marie-France Poupon  1 , Marc-Henri Stern  9   10 , Ivan Bièche  3 , Yves Pommier  17 , Elisabetta Marangoni  18
Affiliations

BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers

Florence Coussy et al. Sci Transl Med. .

Abstract

Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.

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Conflict of interest statement

Competing interests: Y.P. is an inventor on NIH patents for LMP400 and LMP776: US 8,053,443 B2; US 8,829,022 B2; US 9,217,010 B2; US 9,388,211 B2; US 9,399,660 B2; JP541211382; JP5567157 B2; EP1960366 B1, WO2007059008 A2. The title of the patents is “N-substituted indenoisoquinolines and syntheses thereof.” T.P. and M.-H.S. are coinventors of the LST method for detecting inactivation of the HR pathway (BRCA1/2) in human tumors, patent numbers 20170260588 and 20150140122, under exclusive licensing with Myriad Genetics.

Figures

Fig. 1.
Fig. 1.. Response to irinotecan in TNBC PDXs.
(A) Waterfall plot representing responses to irinotecan treatment in 40 TNBC PDXs. Each bar represents the median change in tumor volume from baseline in treated xenografts; n = 4 to 13 xenografts per group. (B) Irinotecan response in HBCx-39, HBCx-10, and HBCx-4B. Means ± standard deviation, n = 8 to 13. (C) Response rates (number of PDXs) to irinotecan according to patients’ pretreatment. NAC, neoadjuvant chemotherapy. (D) Response rates to irinotecan according to patients’ distant relapse after surgery for the primary tumor.
Fig. 2.
Fig. 2.. Response to irinotecan and BRCAness.
(A) Waterfall plot representing irinotecan responses in PDXs with (green) and without BRCAness (gray). BRCA1/2 mutations and BRCA1 methylation are indicated with * and M, respectively. (B) Contingency analysis of BRCA1/2 mutations and BRCAness (Fisher’s exact test). R, response; SD, stable disease; PD, progressive disease; wt, wild type. (C) Response to irinotecan in HBCx-60 (BRCAness) and HBCx-106 (no BRCAness). n = 4 to 5, means ± standard deviation. (D) Western blot analysis of 2AX in HBCx-60 and HBCx-106 xenografts, 4 and 24 hours after a single treatment with irinotecan (n = 3). (E) Percentage of geminin-positive nuclei with more than 10 RAD51 foci in HBCx-106 and HBCx-60 xenografts (control and irinotecan-treated groups, tumors harvested 24 hours after a single treatment); n = 3. ns, not significant. (F) Representative images showing RAD51 foci (green) and geminin (red) immunofluorescence in HBCx-106 and HBCx-60 xenografts harvested 24 hours after a single irinotecan treatment. Scale bars, 10 m. DAPI, 4′,6-diamidino-2-phenylindole.
Fig. 3.
Fig. 3.. Identification of SLFN11 expression and RB1 down-regulation as potential markers of irinotecan response.
(A) Volcano plot displaying differentially expressed genes between responding PDXs (R, n = 15) as compared to nonresponding PDXs (PD, n = 15). The y axis corresponds to log10 (P value), and the x axis displays the log2 fold change value. X-axis grid cutoff lines are shown for fold change of 1.5 and −1.5 and y-axis grid line at P value of 0.05. (B) Robust Multichip Average (RMA)–normalized expression of SLFN11 and RB1 genes. (C) RT-PCR analysis of SLFN11 and RB1 expression. Results are expressed as n-fold differences in target gene expression relative to the TBP gene. (D) SLFN11 expression in HBCx-39 (negative), HBCx-40 (low expression), and HBCx-14 (high expression) analyzed by IHC. Scale bars, 50 m. (E) Pearson’s correlation between SLFN11 gene (RT-PCR) and protein (H-score) expression. r = 0.6992; P < 0.0001 (two-tailed). (F) Frequency distribution of SLFN11 H-scores in the whole set of PDXs. (G) SLFN11 H-scores in PD as compared to R and in PD and SD categories as compared to R.
Fig. 4.
Fig. 4.. Combination markers correlated with irinotecan response.
(A) A waterfall plot showing SLFN11 expression and irinotecan response. (B) Combination of SLFN11 expression and BRCAness as potential markers of response to irinotecan. (C) Contingency analysis, Fisher’s exact test. (D) Individual tumor growth curves of HBCx-60, HBCx-145, HBCx-9, HBCx-15, and HBCx-66 xenografts treated with irinotecan (n = 5 to 10) and olaparib for HBCx-15 and HBCx-66 PDXs (n = 3). (E) SLFN11 expression in HBCx-60, HBCx-145, HBCx-9, HBCx-15, and HBCx-66. Scale bars, 50 m. (F) Waterfall plots showing irinotecan response, RB1 loss determined by IHC, and BRCAness. (G) Contingency analysis, Fisher’s exact test.
Fig. 5.
Fig. 5.. Combination of irinotecan with an ATR inhibitor and antitumor activity of indenoisoquinolines.
(A) Tumor response to irinotecan (40 mg/kg) and the ATR inhibitor VE-822 (VX-870, berzosertib; 50 mg/kg) in the HBCx-1 and HBCx-23 PDXs (BRCAness positive and SLFN11 negative); n = 4 for control, irinotecan, and VE-822–treated groups; n = 7 for the combination group (HBCx-1); and n = 8 for the HBCx-23 xenograft groups. Statistical significance of the difference between irinotecan and irinotecan + VE-822–treated groups was determined by the Mann-Whitney test. (B) Western blot analysis of P-CHK1 (Ser345), P-CHK2, and KU80 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in treated xenografts after a single dose of irinotecan (tumors harvested 24 hours after irinotecan treatment) alone or associated with two injections of VE-822 (administered at days 1 and 2, tumors harvested 4 hours after the second VE-822 treatment); n = 2 for control, irinotecan, and VE-822 xenografts; n = 3 for irinotecan + VE-822 xenografts (HBCx-1); and n = 3 or 4 xenografts for HBCx-23. (C) Antitumor activity of the indenoisoquinolines LMP400 (indotecan) and LMP776 (indimitecan) in the HBCx-60, HBCx-8, HBCx-10, and HBCx-39 PDXs. Means ± standard deviation, n = 5 to 6. RTV, relative tumor volume.
Fig. 6.
Fig. 6.. SLFN11, RB1, and BRCAness and response to AC.
(A) Response to AC and irinotecan in 39 TNBC PDXs (expressed as a fraction of the total tumor number). (B) Combination of SLFN11 expression, RB1 loss, and BRCAness as correlates of AC response in 39 TNBC PDXs. P values were calculated with the Fisher’s exact test. (C) MFS (metastasis-free survival). and OS of 250 patients with TNBC according to SLFN11 gene expression, determined by RT-PCR analysis. Survival distributions were estimated by the Kaplan-Meier method, and P values were calculated with the log-rank test.
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