SLFN11 informs on standard of care and novel treatments in a wide range of cancer models

Abstract

Background: Schlafen 11 (SLFN11) has been linked with response to DNA-damaging agents (DDA) and PARP inhibitors. An in-depth understanding of several aspects of its role as a biomarker in cancer is missing, as is a comprehensive analysis of the clinical significance of SLFN11 as a predictive biomarker to DDA and/or DNA damage-response inhibitor (DDRi) therapies.

Methods: We used a multidisciplinary effort combining specific immunohistochemistry, pharmacology tests, anticancer combination therapies and mechanistic studies to assess SLFN11 as a potential biomarker for stratification of patients treated with several DDA and/or DDRi in the preclinical and clinical setting.

Results: SLFN11 protein associated with both preclinical and patient treatment response to DDA, but not to non-DDA or DDRi therapies, such as WEE1 inhibitor or olaparib in breast cancer. SLFN11-low/absent cancers were identified across different tumour types tested. Combinations of DDA with DDRi targeting the replication-stress response (ATR, CHK1 and WEE1) could re-sensitise SLFN11-absent/low cancer models to the DDA treatment and were effective in upper gastrointestinal and genitourinary malignancies.

Conclusion: SLFN11 informs on the standard of care chemotherapy based on DDA and the effect of selected combinations with ATR, WEE1 or CHK1 inhibitor in a wide range of cancer types and models.

Fig. 1. In vitro SLFN11 correlates with response to DDA and some DDRi. SLFN11 low or absent cancers are found across different tumour indications.

a Correlation of SLFN11 mRNA levels (RMA normalised gene expression) from 738 cell lines with the response to ~589 monotherapy anticancer therapeutics in a multidisciplinary effort from GDSC and AstraZeneca. GDSC genomics of drug sensitivity in cancer. The inset shows the 20 most significantly correlated compounds with SLFN11. b SLFN11 protein in Champions Oncology Patient-Derived Xenograft (PDX) models of the different cancer types as determined by IHC assay (N = 472). c SLFN11 RMA normalised gene expression in cell lines from GDSC grouped by tissue of origin. The bars represent medians ± s.d.

Fig. 2. SLFN11 associates with both preclinical and clinical response to DDA therapies in breast cancer. SLFN11 low cancers are less responsive to DDA treatment.

a Frequency distribution of SLFN11 H-scores in the analysed breast/ovarian cancer cohort. b Pearson’s correlation between SLFN11 gene (RNA-sequencing) and protein (H-score) expression. R = 0.7374; P < 0.0001. c Representative images of nuclear SLFN11 determined by IHC in breast and ovarian PDX models. Left, SLFN11 low and right, SLFN11 high, for the indicated cancers. The arrows indicate mice stromal and endothelial cells that are SLFN11 negative. Scale bars, 100 µm. d Violin plots representing median % TGI for non-DDA and DDA monotherapy treatment in SLFN11 low (<31 H-score) and high (>31 H-score) breast cancer PDX’s (Wilcoxon test). e Box plots depicting median SLFN11 H-scores for non-DDA and DDA treatment in responding (%TGI > 50) and non-responding (%TGI < 50) breast cancer PDX’s. The red continuous line represents the cut point of 31 SLFN11 H-score.

Fig. 3. Resistance to DDA due to absent SLFN11 can be reversed by inhibition of ATR, WEE1 and CHK, but not other DDRi.

a Left, HSA synergy scores of the tested gemcitabine-DDRi combinations in DU145 isogenic cells with the indicated drugs (in triplicates for WT and KO1; n = 1 for KO2). Right, representative heatmaps of combination activity in excess of the calculated HSA model. Prexasertib is the CHKi. b, c Response to gemcitabine in DU145 isogenic cells in the absence or presence of 1 µM ATRi (b) and 0.36 µM WEE1i (c) (continuous treatment for 72 h) as determined by CellTiter-Glo^® luminescent cell viability assays (n = 3). Data are presented as mean percentages ± s.d. (n = 3) of the DMSO and single-agent DDRi-treated conditions.

Fig. 4. SLFN11 protein levels are not decreased following chemotherapy treatment; ATR or WEE1 inhibition restores DNA damage and replication stress in SLFN11-deficient cells.

a Scheme for the protocol used to study SLFN11 protein evolvement following treatment with SN-38 in DU145 wild-type cells as further specified in “Methods”. Also shown are immunoblot analyses of SLFN11 and the indicated biomarkers. *, signal derives from a reprobing of GAPDH blots with pRPA S4/8. b Representative immunoblots of SLFN11 and the indicated biomarkers in DU145 isogenic pair treated for the indicated hours with gemcitabine monotherapy or gemcitabine/ATRi/WEE1i combinations. Similar results were obtained in a second, independent, experiment. *, aspecific bands.

Fig. 5. ATR or WEE1 inhibition induces DNA damage and replication stress in SLFN11-deficient gemcitabine or etoposide-treated cells.

a Staining for DNA (DAPI), pRPA (S4/8) and pH2Ax in DU145 isogenic cells treated for the indicated time points with the designated compounds. The insets show a ×2 magnification. Scale bar, 10 µm. b, c pRPA (S4/8) and pH2Ax mean intensities in DU145 isogenic cells treated with gemcitabine, ATRi (b) or etoposide, ATRi (c) for the indicated time points. The data are presented as mean ± s.e.m. (n = 4) of the control-treated condition. *P < 0.05; **P < 0.01; ***P < 0.001 (paired Student’s t test).

Fig. 6. Resistance to broad DDA in SLFN11 absent/low cancers (different cancer types) can be overcome by inhibition of ATR, WEE1 or CHK1.

a HSA synergy scores of the indicated combinations in SLFN11 high and low pancreatic cancer cell lines. Data are presented as medians with interquartile range (Wilcoxon test). *P < 0.05. b Log IC₅₀ values of cell lines from the indicated cancers following treatments with gemcitabine (GDSC003 dataset) or gemcitabine/ATRi (additional screen, caveat). Data are presented as means ± s.d. *P < 0.05; ****P < 0.0001; ns not significant (one-way ANOVA with Dunnett’s T3 multiple comparisons test). The dashed lines indicate the maximum concentrations of gemcitabine or gemcitabine/ATRi used in the screen. Miscellaneous upper gastrointestinal cancer: HNSC head and neck squamous cell carcinoma, ESCA oesophageal carcinoma. Miscellaneous genitourinary cancer: PRAD prostate adenocarcinoma, BLCA bladder urothelial carcinoma, UCEC uterine corpus endometrial carcinoma, OV ovarian serous cystadenocarcinoma. c Scatter plots of SLFN11 low and high pan-cancer cell lines following 72-h combination treatments. The solid horizontal line indicates the threshold for the maximum activity of the combination and the dashed line the excess effect over the highest single agent (HSA). The black colour indicates cell lines that pass both thresholds and benefit from the combination treatment (indicated as percentages of total cell lines evaluated in the plots). P values derive from a two-sided Fisher’s exact test as further described in the “Methods”. SRA737 is the CHK1i in the left plot.