SLFN11 expression

As single agent, M1774 suppressed cancer cell viability at nanomolar concentrations, showing greater activity than ceralasertib and berzosertib, but less potency than gartisertib and elimusertib in the small-cell lung cancer H146, H82, and DMS114 cell lines. Low dose of M1774 was found highly synergistic with a broad spectrum of clinical DDAs including TOP1 inhibitors (SN-38/irinotecan, topotecan, exatecan, and exatecan), the TOP2 inhibitor etoposide, cisplatin, the RNA polymerase II inhibitor lurbinectedin, and the PARP inhibitor talazoparib in various models including cancer cell lines, patient-derived organoids, and mouse xenograft models. Furthermore, we demonstrate that M1774 reverses chemoresistance to anticancer DDAs in cancer cells lacking SLFN11 expression, suggesting that SLFN11 can be utilized for patient selection in upcoming clinical trials.

Combination of TAK-243 with current ACC therapies (e.g., mitotane, etoposide, cisplatin) produced synergistic or additive effects. In addition, TAK-243 was highly synergistic with BCL2 inhibitors (Navitoclax and Venetoclax) in preclinical ACC models including patient-derived organoids...These findings provide preclinical evidence to support the initiation of a clinical trial of TAK-243 in patients with advanced-stage adrenocortical carcinoma. TAK-243 is a promising potential treatment option for ACC, either as monotherapy or in combination with existing therapies or BCL2 inhibitors.

P2, N=44, Recruiting, ETOP IBCSG Partners Foundation | Not yet recruiting --> Recruiting

P2, N=44, Not yet recruiting, ETOP IBCSG Partners Foundation | Initiation date: Dec 2023 --> Mar 2024

To our knowledge this is the first report of the stable non-lethal increase of SLFN11 expression in a cancer cell line. Our results show that induction of SLFN11 expression can enhance DDA and DDR sensitivity in breast cancer cells and dCas9 systems may represent a novel approach to increase SLFN11 and achieve higher sensitivity to chemotherapeutic agents, improving outcome or decreasing required drug concentrations. SLFN11-targeting therapies might be explored pre-clinically to develop personalized approaches.

Overall, biomarker-focused selection of models led to high efficacy of in vivo treatment. These data enable a paradigm shift to biomarker-driven trial designs for maximizing clinical benefit of ADC therapies.

Importantly, markedly increased sensitivity to cisplatin and SN-38 was seen in initially SLFN11-negative medulloblastoma cells also treated with RG2833, suggesting an approach by which more aggressive medulloblastoma subtypes might be targeted. Our findings suggest a novel mechanism for the increased chemosensitivity of some medulloblastoma subtypes linked to a specific biomarker, as well as a novel combinatorial chemotherapeutic strategy for the treatment of more aggressive medulloblastoma subtypes (Groups 3 and 4) lacking SLFN11.

Based on these results, we propose that mouse Slfn8 and Slfn9 genes may share an orthologous function with human SLFN11. This notion may facilitate understanding of SLFN11's biological role through in vivo studies via mouse modeling.

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.

In metaplastic breast cancers (MpBC), there is limited expression of TROP2, and downregulating transcription factor ZEB1 upregulates E-cad and TROP2, thus sensitizing cancers to TROP2 ADC sacituzumab govitecan (SG). Decitabine also increases SLFN11, a biomarker of topoisomerase 1 inhibitor (TOP1) sensitivity and is synergistic with SG which has a TOP1 payload, in TROP2-expressing SLFN11-low BC cells. In conclusion, TROP2 and SLFN11 expression can be epigenetically modulated and the combination of demethylating agent decitabine with TROP2 ADCs may represent a novel therapeutic approach for tumors with low TROP2 or SLFN11 expression.

We conclude that prognostic and treatment predictive biomarker discovery can improve the management of patients, especially in metastatic stages of advanced PCa. This will result in decreased mortality and enhanced quality of life and help design a personalized treatment regimen.

P2, N=44, Not yet recruiting, ETOP IBCSG Partners Foundation | Initiation date: May 2023 --> Aug 2023

P2, N=24, Active, not recruiting, National Cancer Institute (NCI) | Trial completion date: Jun 2023 --> Apr 2024 | Trial primary completion date: Jun 2023 --> Aug 2022

The aim of this study was to analyze the activity of lurbinectedin in the different SCLC molecular subtypes and to investigate new biomarkers of response.Methods and Results - We have characterized a panel of 20 SCLC human cell lines based on the expression of ASCL1, NEUROD1, YAP1 and POU2F3, where lurbinectedin yielded a mean IC50 value of 6.52 nM, which is considerable greater than the activity exerted by topotecan, irinotecan, carboplatin, etoposide, olaparib, alisertib and navitoclax (IC50 100 µM-100 nM). Finally, SLFN11 expression was evaluated by IHC in FFPE tumor samples from SCLC patients participating in a multicenter phase II clinical trial in advanced solid tumors (NCT02454972), which allowed lurbinectedin accelerated approval in this indication by FDA. Patients with higher expression of SLFN11 had a slightly better overall survival (OS at 6 months: 68.4% (<15%, N=20) vs. 98.7% (≥15%, N=20) p=0.0261)), especially in the refractory/resistant subgroup (OS at 6 months: 33.3% (<15%, N=9) vs. 100.0% (≥15%, N=6) p<0.0001).Conclusions - Lurbinectedin is highly effective in all molecular subtypes of SCLC in vitro and in vivo, with IC50 values at least two logs more potent than for other antitumoral agents and its activity is even greater in tumors with high POU2F3 expression and/or high SLFN11 expression.

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. 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 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.

Genetic and expression alterations identified by the BostonGene Tumor PortraitTM TestEvent type analyzed in targeted panelsEvent typeCohort (N patients)*% cohort with event or # biomarkersEvents, status, or subtypeYesSNV/IndelMG (21)10%FAT4 Y558_V590delinsCAfs*5 LOF mutation COL2A1 W1348Nfs*13 LOF mutationYesSNV/IndelMD (40)6%KMT2B R1779* LOF mutation KMT2C S143Vfs*3 LOF mutation PRKDC Y4046* LOF mutationYesGermlineMG (21)5%MUTYH rs36053993, germline, pathogenicYesGermlineMD (40)3%BTD rs13078881, germline, pathogenicYesCNAMG (21)43%IGF1R amplification +8 copies MGMT loss FANCA loss MCL1 amplification +10 copies MYOCD amplification +3 copies AKT2 amplification +8 copies AMACR amplification +6 copies YAP1 amplification +7 copies TRAF2 loss EZH2 amplification +2 copies HMGA2 amplification +100 copies FRS2 amplification +61 copies DDIT3 amplification +39 copiesYesCNAMD (40)27.5%ERK1 amplification +2 copies FOXO1 amplification +5 copies HMGA2 amplification +2, +8 copies KMT2C amplification +3 copies NCOR1 amplification +7 copies TERT amplification +6 copies TSPAN31 amplification +30 copies HSF1 lossYesFusionsMG (20)10%MDM2-CR1 MDM2-TXNDC12 AC090825.1-IGF1RYesFusionsMD (39)15%FUS-KIAA1549 KIAA1549-CREB3L2 HMGA2-LRRC37A3 NAB2-STAT6 PPP1R12A-PAWR RB1-ZAR1LNoTMB**MG+MD (61)8%35.67 mut/Mb (Desmoid fibromatosis) 8.48 mut/Mb (Cutaneous angiosarcoma) 8.9 mut/Mb (Sarcoma, NOS) 16.1 mut/Mb (Skin Angiosarcoma) 18.8 mut/Mb (Undifferentiated pleomorphic sarcoma)NoMSI statusMG+MD (61)100%StableNoHLA loss of heterozygosityMG+MD (61)11%HLA-I LOH (Leiomyosarcoma (N=1), Ewing Sarcoma (N=1)) HLA-A (Dedifferentiated liposarcoma (N=1)) HLA-I (Chondrosarcoma (N=3), High-grade sarcoma (N=1))NoTargetable surface molecule overexpressionMG+MD (59)81%CTLA4, EGFR, ERBB2, MAGEA3, PD1, PDL1, TIM3, TROP, ERBB2, FOLR1, NECTIN4, ROR1, TROP2NoMolecular Functional PortraitTM typeMG+MD (59)32%FibroticNoMolecular Functional PortraitTM typeMG+MD (59)17%Immune DesertNoMolecular Functional PortraitTM typeMG+MD (59)24%Immune-Enriched, FibroticNoMolecular Functional PortraitTM typeMG+MD (59)27%Immune-Enriched, Non-fibroticNoMHC deficiencyMG+MD (59)3%MHC class I/II deficiencyNoMHC deficiencyMG+MD (59)3%MHC class II deficiencyNoFDA label biomarkersMG+MD (59)7 biomarkers- Desmoid fibromatosis, TMB 35.67 mut/Mb (Pembrolizumab) - Angiosarcoma, TMB 16.1 mut/Mb (Pembrolizumab); - Undifferentiated pleomorphic sarcoma, MSI, TMB 18.8 mut/Mb (Pembrolizumab); - Cutaneous angiosarcoma, PALB2 pathogenic germline variant (Olaparib); - Chondrosarcoma IDH1 R132L (Ivosidenib)NoTranscriptomic biomarkersMG+MD (59)29 biomarkersCD8+ T cell number; PDL1 expression level; SLFN11 expression level; SMARCB1 expression; CD8+ T cell numberNoDiagnostic biomarkersMG+MD (59)9 biomarkersNAB2-STAT6; MXI1-NUTM1; EWSR1-NR4A3; EWSR1-FLI1; EWSR1-CREB3L1; NAB2-STAT6----*RNA analysis failed for 2 patients. Therefore, any expression based analysis was performed on n=20 and n=39 for the MG and MD cohort, respectively. **Tumor mutational burden is considered high for FFPE samples when TMB>8mut/Mb.

Furthermore, SLFN11 high-expressing Hut78 was sensitive to etoposide, a DNA-damaging agent, if compared to other SLFN11 negative cells (HH, MyLa, SeAx). Taken together, our findings suggested that the expression of SLFN11 was closely associated with CD30 expression in MF/SS, that might be regulated via JAK axis. We suppose that SLFN11 might be a predictive biomarker of sensitivity to DNA-damaging agents in patients with CD30-positive MF/SS.

P2, N=44, Not yet recruiting, ETOP IBCSG Partners Foundation

We performed immunohistochemical analyses for SLFN11 expression in 161 HNSCC tissues from patients who had been administered cisplatin-based CRT and examined the correlation between SLFN11 expression and progression-free survival (PFS). Results of the in vitro assay demonstrated that SLFN11-knockout cells exhibited reduced sensitivity to DNA-damaging agents but not to the non-DNA-damaging agent docetaxel. Our findings suggest that SLFN11 may serve as a potential biomarker for predicting the response of HNSCC patients to platinum-based CRT.

Hypomethylating agents (HMAs) such as azacitidine (AZA) and decitabine (DAC) are now frequently used in AML treatment as less intensive treatment options...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...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... 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.

High SLFN11 expression is one factor which renders favorable outcomes in WNT-activated and a subset of SHH-activated medulloblastoma possibly through enhancing response to cisplatin.

Specifically, we covered the combination of PARPi with DNA-damaging chemotherapy (cisplatin, etoposide, temozolomide), thoracic radiotherapy, immunotherapy (immune checkpoint inhibitors), and many other novel therapeutic agents that target DNA damage response, tumor microenvironment, epigenetic modulation, angiogenesis, the ubiquitin-proteasome system, or autophagy. The future of SCLC treatment is undergoing rapid change with a focus on tailored and personalized treatment strategies. Further development of cancer therapy with PARPi will immensely benefit at least a subset of biomarker-defined SCLC patients.

GR-CDXL1 presented homologous recombination deficiency linked to biallelic BRCA2 mutation and FANCA deletion, unrepaired DNA lesions after mitosis, and olaparib sensitivity, despite resistance to chemotherapy...Centrosome clustering promoted targetable chromosomal instability in GR-CDXL3 cells. These CDX unravel DDR and genome integrity-related defects as a central mechanism underpinning metastatic potency of CTCs and provide rationale for their therapeutic targeting in metastatic NSCLC.

P2, N=25, Active, not recruiting, National Cancer Institute (NCI) | Trial completion date: Jun 2022 --> Jun 2023 | Trial primary completion date: Jun 2022 --> Jun 2023

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.

Sacituzumab govitecan (SG), a humanized anti-TROP2 antibody conjugated with SN-38, was approved for treatment of metastatic triple negative breast cancer, with the greatest efficacy in patients with medium or high TROP2 levels. TROP2 is expressed in most breast cancers, but is expressed less frequently in MpBC, an aggressive subtype unresponsive to traditional therapies. Epigenetic modulator decitabine upregulates TROP2 and SLFN11 expression and enhances antitumor efficacy of SG. Combinatorial treatment of TROP2 ADCs with epigenetic modulators of TROP2 represent a novel therapeutic strategy for tumors with low TROP2 or SLFN11 expression.

The antitumor activity of Dato-DXd in combination with PARP inhibition (olaparib) was assessed in 3 PDXs with intermediate Dato-DXd activity. Dato-DXd is a promising therapy for breast cancer patients including those resistant to standard chemotherapy. Additional biomarkers may better integrate DXd sensitivity into patient selection.

This study unravels distinct DDR profiles as a central mechanism underpinning CTC metastatic potency. Our CDX models provide a robust platform for ex vivo drug testing of DDR-targeted strategies to expand patient categories that may benefit from precision medicine in metastatic NSCLC.

Conversely, low SLFN11 expression rendered GSCs susceptible to the oncolytic flavivirus Zika. This discovery effort based upon ASE revealed novel points of vulnerability in GSCs, suggesting a potential alternative treatment strategy for chemotherapy resistant glioblastoma.

The epigenetic modifying drugs 5-azacytidine and entinostat restored SLFN11 expression and re-sensitized 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.

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. 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.

Finally, we assessed the methylation status of the SLFN11 promoter in 27 SCLC clinical specimens, and found that most of the clinical samples (24/27) showed DNA methylation at the SLFN11 promoter. In conclusion, it is feasible to combine topotecan with FK228 to improve the response rate of topotecan in SCLC patients.

In these cells, the clinical JAK inhibitors cerdulatinib, ruxolitinib, and tofacitinib reduced SLFN11 expression, but IFN did not further induce SLFN11 despite phosphorylated STAT1...Accordingly, the AKT and ERK inhibitors MK-2206 and SCH77284 suppressed SLFN11 expression...Moreover, SLFN11 expression was inhibited by the ETS inhibitor TK216. Our study reveals that SLFN11 expression is regulated via the JAK, AKT and ERK, and ETS axis. Pharmacological suppression of SLFN11 warrants future studies.

In addition, enrichment analysis showed that SLFN11 was closely associated with immune-related signalling pathways, and further exploration comprehensively demonstrated strong positive correlations with tumour immune lymphocytes, immune checkpoint genes, chemokines and chemokine receptors. Overall, our data analysis shows that SLFN11 is a strong diagnostic and prognostic biomarker for ccRCC and is also associated with immune infiltration in the TME.

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.

Sensitivity to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN) was assessed in sarcoma cell lines, including SLFN11 knock-out and over-expression models, and a patient-derived orthotopic xenograft model (PDOX). Although SLFN11 appears to drive sensitivity to replicative stress in pediatric sarcomas, its potential to act as a biomarker may be limited to certain tumor backgrounds or contexts. Impaired apoptotic response may be one mechanism of resistance to DDA-induced replicative stress.

We 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.

The RP2D for ceralasertib plus carboplatin was established as ceralasertib 40 mg QD on days 1-2 administered with carboplatin AUC5 every 3 weeks, with pharmacokinetic and pharmacodynamic studies confirming pharmacodynamic modulation and preliminary evidence of antitumor activity observed.

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.

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.