Schlafen 11 Expression Research Articles

Immunohistochemical Evaluation of Schlafen 11 (SLFN11) Expression in Cancer in the Search of Biomarker-Informed Treatment Targets: A Study of 127 Entities Represented by 6658 Tumors.

Schlafen 11 (SLFN11), a DNA/RNA helicase, acts as a regulator of cellular response to replicative stress and irreversibly triggers replication block and cell death. Several preclinical in vitro studies and clinical trials established that SLFN11 expression predicts outcomes in patients with advanced cancer treated with DNA-damaging chemotherapeutics and more recently with poly(ADP-ribose) polymerase inhibitors. SLFN11 expression status remains unknown in many cancer types, especially in mesenchymal tumors. This study evaluated a cohort of well characterized 3808 epithelial and 2850 mesenchymal and neuroectodermal tumors for SLFN11 expression using immunohistochemistry. Nuclear SLFN11 expression was rare in some of the most common carcinomas, for example, hepatocellular (1%), prostatic (2%), colorectal (5%), or breast (16%) cancers. In contrast, other epithelial tumors including mesotheliomas (92%), clear cell renal cell carcinomas (79%), small cell lung cancers (76%), squamous cell carcinomas of the tonsil (89%) and larynx (71%), or ovarian serous carcinomas (69%) were mostly SLFN11-positive. Compared with epithelial malignancies, SLFN11 expression was overall higher in neuroectodermal and mesenchymal tumors. Most positive entities included desmoplastic small round cell tumor (100%), Ewing sarcoma (92%), undifferentiated sarcoma (92%), solitary fibrous tumor (91%), dedifferentiated liposarcoma (89%), synovial sarcoma (86%), and malignant peripheral nerve sheath tumor (85%). Also, this study identifies tumors with potentially worse response to DNA-damaging drugs including antibody drug conjugates due to the absence of SLFN11 expression. Such entities may benefit from alternative treatments or strategies to overcome SLFN11 deficiency-related drug resistance. Our approach and results should serve as a foundation for future biomarker-associated clinical trials.

Schlafen 11 further sensitizes BRCA-deficient cells to PARP inhibitors through single-strand DNA gap accumulation behind replication forks

The preferential response to PARP inhibitors (PARPis) in BRCA-deficient and Schlafen 11 (SLFN11)-expressing ovarian cancers has been documented, yet the underlying molecular mechanisms remain unclear. As the accumulation of single-strand DNA (ssDNA) gaps behind replication forks is key for the lethality effect of PARPis, we investigated the combined effects of SLFN11 expression and BRCA deficiency on PARPi sensitivity and ssDNA gap formation in human cancer cells. PARPis increased chromatin-bound RPA2 and ssDNA gaps in SLFN11-expressing cells and even more in cells with BRCA1 or BRCA2 deficiency. SLFN11 was co-localized with chromatin-bound RPA2 under PARPis treatment, with enhanced recruitment in BRCA2-deficient cells. Notably, the chromatin-bound SLFN11 under PARPis did not block replication, contrary to its function under replication stress. SLFN11 recruitment was attenuated by the inactivation of MRE11. Hence, under PARPi treatment, MRE11 expression and BRCA deficiency lead to ssDNA gaps behind replication forks, where SLFN11 binds and increases their accumulation. As ovarian cancer patients who responded (progression-free survival >2 years) to olaparib maintenance therapy had a significantly higher SLFN11-positivity than short-responders (<6 months), our findings provide a mechanistic understanding of the favorable responses to PARPis in SLFN11-expressing and BRCA-deficient tumors. It highlight the clinical implications of SLFN11.

The expression of SLFN11 is related to the sensitivity of bladder cancer cells to DNA damage agents

BackgroundPutative DNA/RNA helicase Schlafen-11 (SLFN11) was studied for its functions in cancer cells in current years, especially its ability of sensitizing cancer cells to DNA-damaging agents (DDA). However, little is known about the roles it plays in bladder cancer. MethodsBioinformatic tools including GEPIA, UALCAN, MEXPRESS, CTRP, cBioPortal, MethSurv, and Metascape were used. RT-qPCR, CCK-8 assay, and methylation specific PCR were used to verify the results of bioinformatic analysis in bladder cancer cells. ResultsCompared to the normal samples, the expression of SLFN11 was lower in BLCA samples. SLFN11 expression was associated with clinical parameters in BLCA patients, especially histological and molecular subtypes. Besides, its expression correlated to the sensitivity of cancer cells to DDA. Furthermore, the frequency of SLFN11 genetic alterations was low in BLCA patients, and methylationwas significantly negatively related to SLFN11 expression. The mRNA levels of SLFN11 in the bladder cancer cells UMUC-3, T24, and T24-GR were significantly lower than that in the normal SV-HUC-1 cells. SLFN11 expression was positively correlated with bladder cancer cells’ sensitivity to gemcitabine. In addition, partial methylation of SLFN11 was detected in T24-GR, and can be inhibited by 5-Aza. ConclusionsSLFN11 expression was lower in BLCA samples and in vitro bladder cancer cells than in the normal ones. The level of SLFN11 correlated to the sensitivity of cancer cells to DDA. Additionally, the methylation of SLFN11 was negatively related to its expression. It couldbe a potential prognostic biomarker for predicting the therapeutic effect of DNA-damaging agents in patients with bladder cancers.

SLFN11 is a BRCA Independent Biomarker for the Response to Platinum-Based Chemotherapy in High-Grade Serous Ovarian Cancer and Clear Cell Ovarian Carcinoma.

BRCA1/2 mutations are robust biomarkers for platinum-based chemotherapy in epithelial ovarian cancers. However, BRCA1/2 mutations in clear cell ovarian carcinoma (CCC) are less frequent compared with high-grade serous ovarian cancer (HGSC). The discovery of biomarkers that can be applied to CCC is an unmet need in chemotherapy. Schlafen 11 (SLFN11) has attracted attention as a novel sensitizer for DNA-damaging agents including platinum. In this study, we investigated the utility of SLFN11 in HGSC and CCC for platinum-based chemotherapy. SLFN11 expression was analyzed retrospectively by IHC across 326 ovarian cancer samples. The clinicopathologic significance of SLFN11 expression was analyzed across 57 advanced HGSC as a discovery set, 96 advanced HGSC as a validation set, and 57 advanced CCC cases, all of whom received platinum-based chemotherapy. BRCA1/2 mutation was analyzed using targeted-gene sequencing. In the HGSC cohort, the SLFN11-positive and BRCA mutation group showed significantly longer whereas the SLFN11-negative and BRCA wild-type group showed significantly shorter progression-free survival and overall survival. Moreover, SLFN11-positive HGSC shrunk significantly better than SLFN11-negative HGSC after neoadjuvant chemotherapy. Comparable results were obtained with CCC but without consideration of BRCA1/2 mutation due to a small population. Multivariate analysis identified SLFN11 as an independent factor for better survival in HGSC and CCC. The SLFN11-dependent sensitivity to platinum and PARP inhibitors were validated with genetically modified non-HGSC ovarian cancer cell lines. Our study reveals that SLFN11 predicts platinum sensitivity in HGSC and CCC independently of BRCA1/2 mutation status, indicating that SLFN11 assessment can guide treatment selection in HGSC and CCC.

Schlafen 11 (SLFN11) Kills Cancer Cells Undergoing Unscheduled Re-replication.

Schlafen 11 (SLFN11) is an increasingly prominent predictive biomarker and a molecular sensor for a wide range of clinical drugs: topoisomerases, PARP and replication inhibitors, and platinum derivatives. To expand the spectrum of drugs and pathways targeting SLFN11, we ran a high-throughput screen with 1,978 mechanistically annotated, oncology-focused compounds in two isogenic pairs of SLFN11-proficient and -deficient cells (CCRF-CEM and K562). We identified 29 hit compounds that selectively kill SLFN11-proficient cells, including not only previously known DNA-targeting agents, but also the neddylation inhibitor pevonedistat (MLN-4924) and the DNA polymerase α inhibitor AHPN/CD437, which both induced SLFN11 chromatin recruitment. By inactivating cullin-ring E3 ligases, pevonedistat acts as an anticancer agent partly by inducing unscheduled re-replication through supraphysiologic accumulation of CDT1, an essential factor for replication initiation. Unlike the known DNA-targeting agents and AHPN/CD437 that recruit SLFN11 onto chromatin in 4 hours, pevonedistat recruited SLFN11 at late time points (24 hours). While pevonedistat induced unscheduled re-replication in SLFN11-deficient cells after 24 hours, the re-replication was largely blocked in SLFN11-proficient cells. The positive correlation between sensitivity to pevonedistat and SLFN11 expression was also observed in non-isogenic cancer cells in three independent cancer cell databases (NCI-60, CTRP: Cancer Therapeutics Response Portal and GDSC: Genomic of Drug Sensitivity in Cancer). The present study reveals that SLFN11 not only detects stressed replication but also inhibits unscheduled re-replication induced by pevonedistat, thereby enhancing its anticancer efficacy. It also suggests SLFN11 as a potential predictive biomarker for pevonedistat in ongoing and future clinical trials.

Abstract 2145: High SLFN11 expression correlates with sensitivity to lurbinectedin in small cell lung cancer (SCLC) models

Abstract Background: Lurbinectedin monotherapy received accelerated FDA approval for adults with metastatic SCLC with disease progression on or after platinum-based chemotherapy. Lurbinectedin binds to GC-rich areas of gene promoters, blocks RNA-polymerase-II and induces degradation via ubiquitin/proteasome machinery. DNA binding of lurbinectedin results in double stranded DNA breaks and DNA damage that leads to cellular apoptosis. Schlafen 11 (SLFN11), a putative DNA/RNA helicase irreversibly binds to DNA replication forks resulting in replication block and is a predictive biomarker of response to therapeutics that elicit DNA damage including cisplatin, topoisomerase I/II inhibitors and PARP inhibitors. Objective: Evaluate the role of SLFN11 expression in predicting response to lurbinectedin, a DNA damaging agent, in human SCLC cell lines and in vivo models. Methods: Cytotoxicity assays: SCLC cell lines DMS 53, DMS 114, NCI-H69, NCI-H82, NCI-H196, NCI-H209, NCI-H211, NCI-H446, NCI-H526, NCI-H841, NCI-H889, NCI-H1048, and SHP-77 were tested with lurbinectedin dose range from 100 nM to 0.01 nM. Cell viability was determined by measuring intracellular ATP to determine percent cellular viability and IC50 of lurbinectedin. SLFN11 expression of 13 SCLC cell lines were retrieved as RNAseq values from the DepMap database. RNAseq value &amp;gt;2 was assigned as SLFN11 high, values &amp;lt;2 assigned as low. Western blot analysis was used to confirm protein expression. The difference in 10log IC50 between SLFN11 high and low lines is determined using Mann-Whitney test. In vivo efficacy studies: High SLFN11 (NCI-H1048) and low SLFN11 (NCI-H889 and NCI-H69) SCLC cell lines were used for xenograft studies in BALB/c nude mice. Mice were randomly allocated to 4 groups (n=8) as tumors reached a volume of 100 - 150 mm3. Groups received either vehicle or lurbinectedin at doses of 0.06, 0.12 or 0.18 mg/kg IV, QWx 3 weeks. Immunohistochemistry was used to assess SLFN11 protein expression on xenograft tissue. Results and Conclusions: Cell viability assays in 13 SCLC models confirm high SLFN11 expressing cell lines are 4-fold more sensitive to lurbinectedin compared to low SLFN11 expressing cell lines (ΔpIC50 = 0.64; p = 0.0451). The in vivo efficacy data confirms that the SLFN11 high NCI-H1048 model is more responsive with 90% TGI compared to 35% observed in SLFN11 low models (NCI-H889 and NCI-H69) at the highest lurbinectedin dose (p-value = 0.006). Efficacy data confirms correlation to SLFN11 protein expression, consistent with the RNA level association. Both in vitro cytotoxicity and in vivo efficacy studies in SCLC models confirm that high SLFN11 expression predicts response to lurbinectedin. Cell viability studies demonstrate that a limited set of SLFN11 low expressing cell lines are sensitive to lurbinectedin, suggesting a role for other genes. Proteomics and genomics work is underway to define additional response biomarkers Citation Format: Aparna Gupta, Kedar S. Vaidya, Janneke J.T.M. Melis, Robert Hauptschein, Graham Brock, Robin Humphreys. High SLFN11 expression correlates with sensitivity to lurbinectedin in small cell lung cancer (SCLC) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2145.

SLFN11 Is a Potential Biomarker and Modulator of Treatment Response in AML

Introduction: Despite recent advances in treatment for acute myeloid leukemia (AML), AML remains a deadly disease, with approximately 70% of patients succumbing to the disease within five years of diagnosis (SEER database 2021). Intensive induction chemotherapy carries a risk of morbidity and mortality, making it a risky treatment for patients who are unlikely to achieve a remission with DNA damaging-based treatments. Hypomethylating agents (HMAs) such as azacitidine (AZA) and decitabine (DAC) are now frequently used in AML treatment as less intensive treatment options. Thus, biomarkers to predict response to DNA damaging agents in AML are needed. We recently found a significant association between AML patient survival and high expression of Schlafen11 (SLFN11), a dynamically inducible key effector of the DNA replication stress response. SLFN11 has been shown to be associated with response to certain chemotherapy agents in some solid tumors in vitro and in vivo (Conteduca et. al. JCO 2019; Shee et. al. PLoS One 2019), but its importance in leukemia has not been well defined. It is also unknown whether SLFN11 levels affect response to HMAs. We hypothesized that SLFN11 can be used as a predictive biomarker for treatment response to DNA-damaging agents in AML but that response to HMAs would be independent of HMAs. We also hypothesized that identifying pathways upregulated in SLFN11-low cells could lead to novel combination therapies to increase sensitivity to chemotherapeutics. Methods: The human leukemia cell lines HEL and U937 cells were used for these studies. U937- and HEL-SLFN11 knockout (SLFN11KO) cells were generated using CRISPR-Cas9 gene editing technology. Cell viability was assessed by WST-1 reagent assay. SLFN11 and other protein levels were measured by Western blotting. Results: SLFN11 protein expression was readily observed by immunoblotting of HEL and U937 cell lysates. U937- and HEL-SLFN11KO cells were incubated with increasing concentrations of cytarabine (AraC), which is the backbone of most chemotherapy regimens currently used in AML treatment. Both U937- and HEL-SLFN11KO cells were more resistant to AraC than their respective control cells. To examine the mechanism of resistance in U937-SLFN11KO cells, SLFN11KO and control cells were treated with AraC for up to 8 hrs, and lysates were subjected to immunoblotting for various DNA damage response proteins. There were higher baseline levels of multiple DNA damage-related proteins (including Chk1 and Chk2) in the SLFN11KO cells. There was also prolonged activation of the DNA damage pathway in U937-SLFN11KO cells, as evidenced by phosphorylation of the ATR substrate Chk1 (Ser345) through 8 hours, whereas phospho-Chk1 levels decreased in control cells by 8 hours. Interestingly, this prolonged activation occurred without a concomitant increase in γH2AX (a marker of DNA damage and double strand breaks). Because U937-SLFN11KO cells demonstrated a more pronounced activation of the ATR/Chk1 pathway in response to AraC, we subjected U937-SLFN11KO and control cells to increasing concentrations of AraC in combination with a small dose (0.3 μM) of the ATR inhibitor VE822. Notably, ATR inhibition restored sensitivity of SLFN11KO cells to AraC. These results suggest that SLFN11KO cells are resistant to DNA damaging agents via upregulation and activation of DNA repair proteins, and that adding inhibitors of these proteins can decrease resistance to chemotherapy agents in SLFN11KO cells. In contrast, SLFN11KO cells remained sensitive to AZA and DAC, suggesting that HMAs may be preferable to AraC for patients with SLFN11-low AML. Conclusions: Low SLFN11 levels predict poor response to standard chemotherapy agents (i.e. AraC) in AML cells, suggesting that SLFN11 may prove to be an important predictive biomarker for AML patients. In addition, SLFN11-low AML cells have an increased reliance on DNA repair pathways, and inhibition of DNA repair proteins such as ATR may enhance responses to AraC in these cells. This raises the possibility of patient selection based on SLFN11 levels for specific combination approaches for the treatment of AML. Figure 1. Kaplan Meier survival curves of AML patients in the TCGA (LAML) database with high (top 50th percentile) or low (bottom 50th percentile) SLFN11 expression using UCSC Xena (Goldman et. al. Nat Biotechnol 2020). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

1712P Usefulness of schlafen-11 expression level in cstage II/III esophageal squamous cell carcinoma

Schlafen-11 (SLFN-11) was recently identified as a predictive marker of platinum-based drugs. In Japan, neoadjuvant chemotherapy, consists of 5-fluorouracil and cisplatin (NAC-FP), followed by surgery is the standard therapy for patients (pts) with resectable esophageal squamous cell carcinoma (ESCC). However, the correlation between SLFN-11 expression level and prognosis in ESCC pts treated with NAC-FP is unclear.

Abstract 5467: NTX301 targets platinum resistant ovarian cancer

Abstract Background: The majority of patients with ovarian cancer (OC) experience tumor relapse and develop chemoresistant tumors, which are fatal. Epigenetic alterations, especially DNA methylation, result in silencing of tumor suppressor and differentiation-associated genes, confer chemoresistance and are linked to stemness. Although our and others’ previous studies demonstrated that hypomethylating agents (HMA) could re-sensitize OC cells to chemotherapy, translation to the clinic has been slow due the poor stability and bioavailability of existing HMAs. Novel agents are critically needed. Methods: NTX 301 is a novel, highly potent and orally bioavailable HMA, in early clinical development, provided by PinotBio. Here, we assessed its anti-tumor effects in OC models by using cell proliferation, stemness assays, and RNA sequencing and validation. Results: OC cells (SKOV3, IC50=5.089nM; OVCAR4, IC50=29.68nM; and OVCAR5 IC50=3.664nM) were highly sensitive to NTX301 (p&amp;lt;0.05) compared to immortalized fallopian tube epithelial cells (FT-190) (IC50=103.3nM). Treatment with NTX301 induced more significant downregulation of the DNA methyltransferases (DNMTs) 1-3 expression in SKOV3 and OVCAR5 cells compared with decitabine (p&amp;lt;0.05). Treatment with low dose NTX301 (100nM) reset the transcriptome of OC cells, inducing about 15,000 differentially expressed genes (DEGs) compared to DMSO (P&amp;lt;0.05). Gene Ontology Enrichment analysis classified upregulated DEGs enriched in biological processes related to cellular response to DNA damage stimulus, DNA repair, positive regulation of cell cycle process; down-regulated DEGs enriched in processes of regulation of alcohol, cholesterol, and fatty acid biosynthesis and regulation of cell migration. GO analysis indicated NTX301 down-regulated molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase activity, known features of cancer stem cells (CSCs). Indeed, treatment with low dose NTX301 (100nM, 4 days) reduced ALDH(+) cell population, inhibited their self-renewal ability, and repressed the stemness-related transcription factor, Sox2 (P&amp;lt;0.05). We next tested the effects NTX301 in cisplatin (CDDP) resistant OC cells, developed by repeated treatments with CDDP. Priming with NTX301 reduced the IC50 (~two-fold) to CDDP in OVCAR5_CDDP (5.43μM vs. 9.79μM) and OVCAR4_CDDP (2.19μM vs. 9.29μM) resistant cells (p&amp;lt;0.05). NTX301 with CDDP induced phospho-γH2AX and cleaved caspase 3. To discover the mechanisms by which NTX301 restored chemosensitivity, we measured its effects on Schlafen-11 (SLFN11), a DNA damage response regulator silenced through methylation. SLFN11 expression was repressed in CDDP resistant compared to parental cells (P&amp;lt;0.05) and treatment with NTX301 induced its re-expression (P&amp;lt;0.05). Conclusions: Our data indicate that NTX301 is a potent HMA which targets ovarian CSCs and re-sensitizes OC cells to chemotherapy, supporting its further development in OC. Citation Format: Yinu Wang, Xiaolei Situ, Horacio Cardenas, Daniela Matei. NTX301 targets platinum resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5467.

Dynamic expression of Schlafen 11 (SLFN11) in circulating tumour cells as a liquid biomarker in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive malignancy with no established biomarkers. Schlafen 11(SLFN11), a DNA/RNA helicase that sensitises cancer cells to DNA-damaging agents, has emerged as a promising predictive biomarker for several drug classes including platinum and PARP inhibitors. Detection of SLFN11 in circulating tumour cells (CTCs) may provide a valuable alternative to tissue sampling. SLFN11 expression was evaluated in tumour samples and characterised in circulating tumour cells (CTC) longitudinally to determine its potential role as a biomarker of response. Among 196 SCLC tumours, 51% expressed SLFN11 by IHC. In addition, 20/29 extra-thoracic high-grade neuroendocrine tumours expressed SLFN11 expression. In 64 blood samples from 42 SCLC patients, 83% (53/64) of samples had detectable CTCs, and SLFN11-positive CTCs were detected in 55% (29/53). Patients actively receiving platinum treatment had the lowest number of CTCs and a lower percentage of SLFN11-positive CTCs (p = 0.014). Analysis from patients with longitudinal samples suggest a decrease in CTC number and in SLFN11 expression that correlates with clinical response. SLFN11 levels can be monitored in CTCs from SCLC patients using non-invasive liquid biopsies. The ability to detect SLFN11 in CTCs from SCLC patients adds a valuable tool for the detection and longitudinal monitoring of this promising biomarker.

Prognostic impact of Schlafen 11 in bladder cancer patients treated with platinum-based chemotherapy.

ABSTRACTThe utility of Schlafen 11 (SLFN11) expression as a predictive biomarker for platinum‐based chemotherapy has been established for cancers from different histologies. However, the therapeutic relevance of SLFN11 in bladder cancer (BC) is unknown. Here, we examined the clinicopathologic significance of SLFN11 expression across 120 BC cases by immunohistochemistry. We divided the cases into two cohorts, one including 50 patients who received adjuvant or neoadjuvant platinum‐based chemotherapy, and the other including 70 BC patients treated by surgical resection without chemotherapy. In the cohort of 50 BC cases treated with platinum‐based chemotherapy, the SLFN11‐positive group (n = 25) showed significantly better overall survival than the SLFN11‐negative group (n = 25, P = .012). Schlafen 11 expression correlated significantly with the expression of luminal subtype marker GATA3. Multivariate analyses identified SLFN11 expression as an independent prognostic predictor (odds ratio, 0.32; 95% confidence interval, 0.11‐0.91; P = .033). Conversely, in the cohort of 70 BC cases not receiving platinum‐based chemotherapy, the SLFN11‐positive group (n = 29) showed significantly worse overall survival than the SLFN11‐negative group (n = 41, P = .034). In vitro analyses using multiple BC cell lines confirmed that SLFN11 KO rendered cells resistant to cisplatin. The epigenetic modifying drugs 5‐azacytidine and entinostat restored SLFN11 expression and resensitized cells to cisplatin and carboplatin in SLFN11‐negative BC cell lines. We conclude that SLFN11 is a predictive biomarker for BC patients who undergo platinum‐based chemotherapy and that the combination of epigenetic modifiers could rescue refractory BC patients to platinum derivatives by reactivating SLFN11 expression.

Abstract P020: Schlafen 11 (SLFN11) as a predictive biomarker of the response to TAS1553, a novel small molecule ribonucleotide reductase subunit interaction inhibitor

Abstract Background: Ribonucleotide reductase (RNR) plays a crucial role in dNTP biosynthesis, which is required for DNA synthesis and repair, and is thought to be an attractive cancer therapeutic target. However, the precise significance of RNR inhibition remains to be elucidated, since reported RNR inhibitors exhibit limited pharmacological potency and off-target effects. We have developed a highly potent and novel small molecule RNR inhibitor, TAS1553, and reported that TAS1553 disrupted the protein-protein interaction between RNR subunits and exhibited the broad antiproliferative activity against human cancer cells in both in vitro and in vivo via oral administration. Phase-I study is currently ongoing and the identification of a predictive biomarker is essential to maximize clinical benefit of TAS1553. Here, we report a predictive biomarker of the response to TAS1553, and a clinical development strategy with the biomarker use. Material and methods: TAS1553 was synthesized at Taiho Pharmaceutical Co., Ltd. DNA replication stress, apoptosis, caspase activity and cellular growth inhibition induced by TAS1553 were assessed by western blotting, immunofluorescence staining, Caspase-Glo 3/7 assay and CellTiter-Glo® 2.0 assay, respectively. Cells were transfected with 2 nM siRNAs against SLFN11 (siSLFN11) and used for caspase-3/7 activation assay and cell proliferation assay. Results: TAS1553 induced intracellular pChk1, pRPA2 and γH2AX, followed by cleavage of PARP and caspase-3, suggesting that TAS1553 causes massive DNA replication stress. Then, we explored factors, involved in DNA replication stress, to predict response to TAS1553. Cytotoxicity profiling revealed broad antiproliferative activity of TAS1553 against both human hematological and solid cancer cell lines in a dose-​dependent manner (GI50 = 228-4150 nmol/L), and global gene expression profiling revealed that cells with high SLFN11 mRNA expression showed a higher sensitivity to TAS1553. TAS1553 exerted growth inhibitory activity without any cell killing effect against cells with low SLFN11 expression even at a concentration of 10 μmol/L, but cell killing activity against cells with high SLFN11 expression. Depletion of SLFN11 by siSLFN11 treatment in A673 cells which have high SLFN11 expression suppressed cytotoxic effect but not the growth inhibitory effect of TAS1553. Furthermore, we observed suppression of caspase-3/7 activation induced by TAS1553 in A673 cells transfected with siSLFN11. SLFN11 appeared to sensitize cancer cells to TAS1553 via promoting apoptosis. Conclusions: TAS1553, a novel orally available RNR inhibitor, showed potent antitumor activity in preclinical models of both hematological and solid tumors. Thus, TAS1553 could be a promising therapeutic agent for cancer, and SLFN11 could be a predictive biomarker in order to maximize clinical response to TAS1553. Citation Format: Hiroto Fukushima, Hiroyuki Ueno, Takuya Hoshino, Wakako Yano, Hiraku Itadani, Miki Terasaka, Sayaka Tsukioka, Takamasa Suzuki, Shoki Hara, Yoshio Ogino, Khoon Tee Chong, Tatsuya Suzuki, Yoshihiro Otsu, Satoshi Ito, Nozomu Tanaka, Seiji Miyahara. Schlafen 11 (SLFN11) as a predictive biomarker of the response to TAS1553, a novel small molecule ribonucleotide reductase subunit interaction inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P020.

SLFN11 biomarker status predicts response to lurbinectedin as a single agent and in combination with ATR inhibition in small cell lung cancer.

BackgroundLurbinectedin recently received FDA accelerated approval as a second line treatment option for metastatic small cell lung cancer (SCLC). However, there are currently no established biomarkers to predict SCLC sensitivity or resistance to lurbinectedin or preclinical studies to guide rational combinations.MethodsDrug sensitivity was assayed in proliferation assays and xenograft models. Baseline proteomic profiling was performed by reverse-phase protein array. Lurbinectedin-induced changes in intracellular signaling pathways were assayed by Western blot.ResultsAmong 21 human SCLC cell lines, cytotoxicity was observed following lurbinectedin treatment at a low dose (median IC50 0.46 nM, range, 0.06–1.83 nM). Notably, cell lines with high expression of Schlafen-11 (SLFN11) protein, a promising biomarker of response to other DNA damaging agents (e.g., chemotherapy, PARP inhibitors), were more sensitive to single-agent lurbinectedin (FC =3.2, P=0.005). SLFN11 was validated as a biomarker of sensitivity to lurbinectedin using siRNA knockdown and in xenografts representing SLFN11 high and low SCLC. Replication stress and DNA damage markers (e.g., γH2AX, phosphorylated CHK1, phosphorylated RPA32) increased in SCLC cell lines following treatment with lurbinectedin. Lurbinectedin also induced PD-L1 expression via cGAS-STING pathway activation. Finally, the combination of lurbinectedin with the ataxia telangiectasia and Rad3-related protein (ATR) inhibitors ceralasertib and berzosertib showed a greater than additive effect in SLFN11-low models.ConclusionsTogether our data confirm the activity of lurbinectedin across a large cohort of SCLC models and identify SLFN11 as a top candidate biomarker for lurbinectedin sensitivity. In SLFN11-low SCLC cell lines which are relatively resistance to lurbinectedin, the addition of an ATR inhibitor to lurbinectedin re-sensitized otherwise resistant cells, confirming previous observations that SLFN11 is a master regulator of DNA damage response independent of ATR, and the absence of SLFN11 leads to synthetic lethality with ATR inhibition. This study provides a rationale for lurbinectedin in combination with ATR inhibitors to overcome resistance in SCLC with low SLFN11 expression.

SLFN11 captures cancer-immunity interactions associated with platinum sensitivity in high-grade serous ovarian cancer.

Large independent analyses on cancer cell lines followed by functional studies have identified Schlafen 11 (SLFN11), a putative helicase, as the strongest predictor of sensitivity to DNA-damaging agents (DDAs), including platinum. However, its role as a prognostic biomarker is undefined, partially due to the lack of validated methods to score SLFN11 in human tissues. Here, we implemented a pipeline to quantify SLFN11 in human cancer samples. By analyzing a cohort of high-grade serous ovarian carcinoma (HGSOC) specimens before platinum-based chemotherapy treatment, we show, for the first time to our knowledge, that SLFN11 density in both the neoplastic and microenvironmental components was independently associated with favorable outcome. We observed SLFN11 expression in both infiltrating innate and adaptive immune cells, and analyses in a second, independent, cohort revealed that SLFN11 was associated with immune activation in HGSOC. We found that platinum treatments activated immune-related pathways in ovarian cancer cells in an SLFN11-dependent manner, representative of tumor-immune transactivation. Moreover, SLFN11 expression was induced in activated, isolated immune cell subpopulations, hinting that SLFN11 in the immune compartment may be an indicator of immune transactivation. In summary, we propose SLFN11 is a dual biomarker capturing simultaneously interconnected immunological and cancer cell–intrinsic functional dispositions associated with sensitivity to DDA treatment.

PARP inhibition in UV-associated angiosarcoma preclinical models

PurposeAngiosarcoma (AS) is a rare vasoformative sarcoma, with poor overall survival and a high need for novel treatment options. Clinically, AS consists of different subtypes, including AS related to previous UV exposure (UV AS) which could indicate susceptibility to DNA damage repair inhibition. We, therefore, investigated the presence of biomarkers PARP1 (poly(ADP-ribose)polymerase-1) and Schlafen-11 (SLFN11) in UV AS. Based on experiences in other sarcomas, we examined (combination) treatment of PARP inhibitor (PARPi) olaparib and temozolomide (TMZ) in UV AS cell lines.MethodsPreviously collected UV AS (n = 47) and non-UV AS (n = 96) patient samples and two UV AS cell lines (MO-LAS and AS-M) were immunohistochemically assessed for PARP1 and SLFN11 expression. Both cell lines were treated with single agents PARPi olaparib and TMZ, and the combination treatment. Next, cell viability and treatment synergy were analyzed. In addition, effects on apoptosis and DNA damage were examined.ResultsIn 46/47 UV AS samples (98%), PARP1 expression was present. SLFN11 was expressed in 80% (37/46) of cases. Olaparib and TMZ combination treatment was synergistic in both cell lines, with significantly increased apoptosis compared to single agent treatment. Furthermore, a significant increase in DNA damage marker γH2AX was present in both cell lines after combination therapy.ConclusionWe showed combination treatment of olaparib with TMZ was synergistic in UV AS cell lines. Expression of PARP1 and SLFN11 was present in the majority of UV AS tumor samples. Together, these results suggest combination treatment of olaparib and TMZ is a potential novel AS subtype-specific treatment option for UV AS patients.

Schlafen 11 (SLFN11), a putative predictive biomarker of platinum/PARPi response, is frequently detected on circulating tumor cells (CTCs) in advanced prostate cancer.

e17039 Background: Schlafen 11 (SLFN11) is a DNA repair protein (DNA/RNA helicase homology) that is recruited to stressed replication forks and leads to cell death. Recent ph II trial data in Extensive Stage Small-Cell Lung Cancer (ES-SCLC) [Byers et al. JCO 2018 PMID: 29906251] and a retrospective analysis of Circulating Tumor Cells (CTCs) and tumor tissue in patients (Pts) with advanced prostate cancer [Conteduca et al. Mol Cancer Therapeutics 2020 PMID: 32127465] suggested that SLFN11 expression predicts sensitivity to DNA damage targeting agents. In both contexts, metastatic tumor biopsies may not provide adequate material for profiling. We assessed the frequency of SLFN11 expression in CTCs isolated from blood in men with progressing metastatic Castration Resistant Prostate Cancer (mCRPC) and related expression to Homologous Recombination Repair (HRR) alterations identified in metastatic tumor biopsies profiled by MSK-IMPACT. Methods: 95 patients with progressing mCRPC about to start a new systemic therapy who had undergone pre-treatment metastatic tumor profiling by MSK-IMPACT and a matched blood draw for CTC profiling were selected. Blood was sent overnight to Epic Sciences and processed onto glass pathology slides and bio-banked until analysis. Detected CTCs (cytokeratin (CK) positive and leukocyte (CD45) negative) were analyzed for SLFN11 protein expression by immunofluorescence and correlated to Homologous Recombination Repair (HRR) alterations in metastatic biopsy from the bone, lymph node, or visceral metastases (15 PROFOUND genes). A mean of 1.2 mL of blood was analyzed per patient. Results: CTCs with SLFN11 expression (DAPI+, CK+, CD45-, SLFN11+) were detected in 28.4% (27/95) of the patient sample analyzed. SLFN11 signal was found to overlap with the nuclear DAPI signal. Individual CTC expression of SLFN11 in a sample was heterogeneous and ranged from a minimum of 2.9% to 100%. Seven of 10 (70%) of patients with a BRCA1/2 or ATM alteration had a least one SLFN11 expressing CTC. In contrast, in patients with other HRR alterations, only 20% (1/5) had CTCs with SLFN11. Sequencing of single CTCs is ongoing. Conclusions: SLFN11 expression is detected with high frequency in CTCs in men with progressing mCRPC. In whom CTCs are detected, the majority of patients with BRCA1/2 or ATM altered tumors also had SLFN11 expressing CTCs. The results support the prospective evaluation of CTC SLFN11 expression as a predictive biomarker for PARPi or platinum agents.

Schlafen 11 predicts response to platinum-based chemotherapy in gastric cancers.

Although unresectable or recurrent gastric cancers (GC) are frequently treated with platinum-based chemotherapy, response to treatment remains unpredictable. Because Schlafen 11 (SLFN11) is recently identified as a critical determinant of platinum sensitivity, we investigated the potential clinical utility of SLFN11 in the treatment of GC. We analysed the correlation between SLFN11 expression and overall survival in 169 GC patients by our established immunohistochemical approach. The impact of SLFN11 expression on the response to platinum and transition of SLFN11 expression upon long-term treatment with platinum were examined using GC cell lines and organoids. GC patients with high-SLFN11 expression exhibited significantly better survival than those with low-SLFN11 expression, and the significance increased when we selected patients treated with platinum-based chemotherapy. Knockout of SLFN11 and reactivation of SLFN11 in GC cells conferred resistance and sensitivity to platinum, respectively. In GC cells and organoids, long-term treatment with oxaliplatin suppressed SLFN11 expression while imparting drug resistance. The acquired resistance to oxaliplatin was reversed by reactivation of SLFN11 with epigenetic modifying drugs. This is the first report revealing definitive clinical implications of SLFN11 in the treatment of GC patients and providing novel strategies for the drug selection based on SLFN11 expression.

Abstract LB-121: The novel ATR inhibitor M4344 and CHK1 inhibitor SRA737 overcome chemoresistance in SLFN11-negative cells in combination treatment with DNA-damaging agents

Abstract Precision medicine is an unmet need for DNA-damaging agents with potentially severe side effects. Loss of Schlafen 11 (SLFN11) expression is frequently detected in ~50% cancer cell lines of the NCI, the Broad Institute cancer cell line panel (CCLE), and the Genomics of Drug Sensitivity in Cancer project (GDSC). Deficiency of SLFN11 expression causes chemoresistance to a broad range of DNA-damaging agents, including topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, alkylating agents, DNA synthesis inhibitors and PARP inhibitors. Moreover, gene silencing of SLFN11 is observed in small cell lung cancer patient-derived xenograft models with acquired drug resistance. To identify synthetic lethal therapeutic targets to overcome chemoresistance in SLFN11 deficient cells, we performed a genome-wide RNAi screen with the human druggable genome siRNA library by using camptothecin (CPT), a TOP1 inhibitor, in SLFN11 wild-type (WT) and knock-out (KO) prostate cancer DU145 cells. Gene Ontology analysis identified the inhibition of ATR-mediated DNA repair pathway genes (ATR, CHK1, BRCA2 and RPA1) is synergistic with CPT in SLFN11 KO cells, whereas inhibition of the RNA metabolism-related genes (POLR2, PSMD3, POLR2F and PSMD11) had higher combination effects in SLFN11 WT cells. The synergistic effects by depletion of ATR-mediated DNA repair pathway in SLFN11 KO cells were validated by additional siRNAs-mediated cell viability assays. To determine whether inhibition of ATR-mediated pathway can be applied clinically, we tested the new clinical ATR (M4344) and CHK1 (SRA737) inhibitors. Treatment with non-toxic-doses of M4344 and SRA737 reversed drug resistance of the SLFN11 KO cells to TOP1 inhibitors [CPT, and clinically used topotecan and LMP400 (indotecan)]. Additionally, the same synergistic effects by combination of the ATR/CHK1inhibitors and CPT were detected in isogenic CCRF-CEM SLFN11 WT and KO leukemic lymphoblasts cells and non-isogenic DMS114 and H446 small cell lung cancer cells. We also confirmed synergy with ATR/CHK1 inhibitors in combination of other clinical DNA-damaging agents (TOP2 inhibitor: etoposide, alkylating agent: cisplatin, and PARP inhibitor: talazoparib). Molecular changes induced by the combination treatment were examined in the cell cycle by assessing DAPI and EdU incorporation, cell death with Annexin V, and DNA damage response by confocal microscopy with M4344 and CPT. Co-treatment with ATR inhibitor and CPT resulted in G2/M arrest and apoptotic cell death, and formation of micronuclei and fragmented nuclei in SLFN11 KO cells, compared with SLFN11 WT cells. Collectively, our results provide a new therapeutic rationale for the clinical development of combination treatments of chemotherapeutic DNA-targeted agents with ATR/CHK1 inhibitors based on SLFN11 status. Citation Format: Ukhyun Jo, Yasuhisa Murai, Junko Murai, Shar-yin N Huang, Sirisha Chakka, Lu Chen, Ken Cheng, Yves Pommier. The novel ATR inhibitor M4344 and CHK1 inhibitor SRA737 overcome chemoresistance in SLFN11-negative cells in combination treatment with DNA-damaging agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-121.

Olaparib and temozolomide in desmoplastic small round cell tumors: a promising combination in vitro and in vivo

PurposeDesmoplastic small round cell tumors (DSRCTs) are highly malignant and very rare soft tissue sarcomas with a high unmet need for new therapeutic options. Therefore, we examined poly(ADP-ribose) polymerase 1 (PARP1) and Schlafen-11 (SLFN11) expression in DSRCT tumor tissue and the combination of PARP inhibitor olaparib with the alkylating agent temozolomide (TMZ) in a preclinical DSRCT model.MethodsPARP1 and SLFN11 have been described as predictive biomarkers for response to PARP inhibition. Expression of PARP1 and SLFN11 was assessed in 16 and 12 DSRCT tumor tissue samples, respectively. Effects of single-agent olaparib, and olaparib and TMZ combination treatment were examined using the preclinical JN-DSRCT-1 model. In vitro, single-agent and combination treatment effects on cell viability, the cell cycle, DNA damage and apoptosis were examined. Olaparib and TMZ combination treatment was also assessed in vivo.ResultsPARP1 and SLFN11 expression was observed in 100% and 92% of DSRCT tumor tissues, respectively. Olaparib treatment reduced cell viability and cell migration in a dose-dependent manner in vitro. Drug synergy between olaparib and TMZ was observed in vitro and in vivo. Combination treatment led to a cell-cycle arrest and induction of DNA damage and apoptosis, even when combined at low dosages.ConclusionWe show high PARP1 and SLFN11 expression in DSRCT tumor material and antitumor effects following olaparib and TMZ combination treatment in a preclinical DSRCT model. This suggests that olaparib and TMZ combination treatment could be a potential treatment option for DSRCTs.

Sensitivity of Mesothelioma Cells to PARP Inhibitors Is Not Dependent on BAP1 but Is Enhanced by Temozolomide in Cells With High-Schlafen 11 and Low-O6-methylguanine-DNA Methyltransferase Expression.

BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase thought to be involved in DNA double-strand break repair, is frequently mutated in mesothelioma. Because poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesothelioma and if combination therapy with temozolomide (TMZ) would be beneficial. A total of 10 patient-derived mesothelioma celllines were generated and characterized for BAP1 mutation status, protein expression, nuclear localization, and sensitivity to the PARPIs, olaparib, and talazoparib, alone orin combination with TMZ. BAP1 deubiquitinase (DUB) activity was evaluated by ubiquitin with 7-amido-4-methylcoumarin assay. BAP1 knockout mesothelioma cell lines were generated by CRISPR-Cas9. Because Schlafen 11 (SLFN11) and O6-methylguanine-DNA methyltransferase also drive response to TMZ and PARPIs, we tested their expression and relationship with drug response. BAP1 mutations or copy-number alterations, or both were present in all 10 cell lines. Nonetheless, four cell lines exhibited intact DUB activity and two had nuclear BAP1 localization. Half maximal-inhibitory concentrations of olaparib and talazoparib ranged from 4.8 μM to greater than 50 μM and 0.039 μM to greater than 5 μM, respectively, classifying them into sensitive (two) or resistant (seven) cells, independent of their BAP1 status. Cell lines with BAP1 knockout resulted in the loss of BAP1 DUB activity but did not increase sensitivity to talazoparib. Response to PARPI tended to be associated with high SLFN11 expression, and combination with temozolomide increased sensitivity of cells with low or no MGMT expression. BAP1 status does not determine sensitivity to PARPIs in patient-derived mesothelioma cell lines. Combination of PARPI with TMZ may be beneficial for patients whose tumors have high SLFN11 and low or no MGMT expression.