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PARP inhibitors (PARPi) have changed the management of patients with ovarian cancer and their effectiveness has been demonstrated especially in homologous recombination repair–deficient tumors. These first-generation drugs target PARP1, but also PARP2 and other family members potentially responsible for adverse effects that limit their therapeutic potential and restrict their use in combination with chemotherapeutic agents. We investigated ovarian cancer patient-derived xenografts (OC-PDXs) to assess whether malignant progression could be impaired by a novel inhibitor selective for PARP1 (AZD5305) and to assess the potential of its combination with carboplatin (CPT), the standard-of-care for patients with ovarian cancer. In BRCA-mutated OC-PDXs, AZD5305 achieved greater tumor regressions and longer duration of response as well as a superior impairment of visceral metastasis and improved survival benefit compared with the first-generation dual PARP1/2 inhibitors. The combination of AZD5305 plus CPT was more efficacious than single agents. Subcutaneously growing tumors experienced regression that persisted after therapy stopped. Combination efficacy was greater against tumors that did not respond well to platinum, even at a dose at which AZD5305 monotherapy was ineffective. The combination therapy impaired metastatic dissemination and significantly prolonged the lifespan of mice bearing OC-PDXs in their abdomen. This combination benefit was evident even when CPT was used at suboptimal doses, and was superior to full-dose platinum treatment. These preclinical studies demonstrate that the PARP1-selective inhibitor AZD5305 retains and improves the therapeutic benefit of the first-generation PARPi, providing an opportunity to maximize benefits for this class of anticancer agents. Significance: Selective PARP1i AZD5305 can exceed the efficacy of first-generation PARPi, which target both PARP1 and PARP2, and potentiates the efficacy of CPT when given in combination. AZD5305 alone or in combination with platinum delayed visceral metastasis, ultimately extending the lifespan of OC-PDX–bearing mice. These preclinical models mimic the progression of the disease occurring in patients after debulking surgery, and are translationally relevant.

Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor approved for HER2+ metastatic breast and gastric cancer. Clinically, T-DXd has demonstrated antitumor activity in both HER2+ and HER2-low cancers. PARP performs a key role as a mediator in the resolution of topoisomerase1 cleavage complexes (TOP1cc) through recruitment of tyrosyl-DNA phosphodiesterase 1 (TDP1). We hypothesized that combination of T-DXd with the PARP1/2 inhibitor olaparib, will halt the resolution of TOP1cc and enhance the activity of T-DXd. Methods: To test the hypothesis, we evaluated the antiproliferative ability of the combination of T-DXd with olaparib in a panel of 27 breast cancer cell lines in an in vitro 7-day viability assay. The combination was also evaluated in vivo in two non-HRD models, a HER2+ (KPL4) and HER2-low (JIMT1) cell line xenograft at 3mg/kg and 10mg/kg Q3W for T-DXd, as well as 100mg/kg BID of olaparib. To evaluate the specificity of the combination activity in tumor cells (vs normal tissue), we further evaluated the combination in a human 2D in vitro bone marrow progenitor assay. Results: We found that the combination had enhanced in vitro cell killing activity over single agents in 8/27 of the models tested. The benefit was present in both Homologous Repair Deficient (HRD) as well as wild-type models, suggesting it does not depend on HRD (as defined by mutations in DNA damage repair genes). In vivo, the combination was more active than monotherapy of either compound in both KPL4 (28d TGI of 76% with T-DXd, olaparib 17% TGI, and T-DXd + olaparib 94.4% TGI; p=0.009) and JIMT1 (28d TGI of 80.8% with T-DXd, olaparib 52%, and T-DXd + Olaparib 88.1%; p=0.03). In the in vitro human bone marrow assay, the combination demonstrated modest enhancement over monotherapy activity (average Loewe Synergy Score of 2.5). To explore the ability to optimize therapeutic index, we tested alternative doses and schedules of the combination in vivo. Specifically, we tested whether lower doses of T-DXd or delayed administration of olaparib would provide greater activity. We found that combination of 3mg/kg of T-DXd with olaparib provided greater activity in KPL4 xenograft model than 3mg/kg T-DXd alone (69% TGI for combination vs. 35% TGI for monotherapy on d28), but this was no greater than high dose 10mg/kg monotherapy T-DXd (76% TGI on d28; p=0.19). Interestingly, 7-day delay of olaparib in combination with 10mg/kg T-DXd provided greater activity (91% TGI on d28) than monotherapy T-DXd or olaparib alone (76% TGI and 17% TGI on d28, respectively). Conclusions: These results suggest that T-DXd combined with olaparib is a potentially active combination in breast cancer, with preclinical activity demonstrated in HRD and non-HRD models. Citation Format: Theresa Proia, Yann Wallez, Azadeh Cheraghchi Bashi, Zena Wilson, Suzanne Randle, Mark Anderton, Danielle Carroll, Zeshaan Rasheed, J. Elizabeth Pease, Elisabetta Leo, Jerome Mettetal. Activity and tolerability of combination of trastuzumab deruxtecan with olaparib in preclinical HER2+ and HER2-low breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-18.

Supplementary Figure S1. A, Workflow to generate isogenic HRR KO cell models (see also Methods). B, Dose-response curve for SKOV3 BRCA2 KO isogenic pairs treated with olaparib for 10-14 days in clonogenic survival assays. Results are shown as mean of n=4 biological replicates ± SD for the dose-response curves. Clone 13 was selected for further experimentation. C, Fold-change mRNA expression in WT and KO cells to assess BRCA2 loss in SKOV3 cells. D, Dose-response curve for DU145 BRCA1 KO isogenic pairs treated with olaparib for 10-14 days in clonogenic survival assays. Results are shown as mean of n=2 biological replicates ± SD for the dose-response curves. Clone A1 was selected for further experimentation. E, Western blot data to assess BRCA1 loss in DU145 cells (clone A1). Supplementary Figure S2. A, Dose-response curve for SKOV3 PALB2 KO isogenic pairs treated with olaparib for 10-14 days in clonogenic survival assays. Results are shown as mean of n=4 biological replicates ± SD for the dose-response curves. Clone 19 was selected for further experimentation. B, Western blot data to assess PALB2 loss in SKOV3 cells (clone 19). C, Dose-response curve for SKOV3 RAD51C KO clone 3 isogenic pairs treated with olaparib for 10-14 days in clonogenic survival assays. Results are shown as mean of n=3 biological replicates ± SD for the dose-response curves. D, Western blot data to assess RAD51C loss in SKOV3 cells (clone C7 was selected for further experimentation and its dose-response curve is shown in Figure 2A). E, Dose-response curve for SKOV3 RAD51B KO isogenic pairs treated with olaparib for 10-14 days in clonogenic survival assays. Results are shown as mean of n=4 biological replicates ± SD for the dose-response curves. Clone 4 was selected for further experimentation. F-H, Fold-change mRNA expression in WT and KO cells to assess RAD51B loss in SKOV3 cells (clone 4) (F) and DU145 cells (G), and loss of RAD54L in DU145 cells (H). Supplementary Figure S3. A-E and G, Western blots to assess ATM loss (A, C) and ATM signalling (B, D, E, G) 4 h after treating cells with 5 Gy of ionizing radiation (IR) in the presence or absence of ATM inhibitor (AZD0156, 100 nM) added 2 h before treatment in DU145 (B), SKOV3 (D), DLD1 (E) and LNCAP (G) ATM KO isogenic cell lines. F and H, Dose-response curves for ATM KO DLD1 clones (F) and LNCAP (H) isogenic pairs treated with olaparib for 10-14 days in clonogenic survival assays. Results are shown as mean of n=4 biological replicates ± SD for the dose-response curves. Clonogenic survival data on (H) was generated with LNCAP ATM KO2 cells (see panel G). Clone 5 of the DLD1 ATM KO cells was selected for further experimentation. Supplementary Figure S4. A, Pan-cancer analysis of the total number of samples showing biallelic loss of the 110 confidence genes identified through CRISPR screens. B, Oncoprint of the top 10 genes showing biallelic LoF in the pan-cancer analysis. Each bar represents an individual tumour. Percentages are for the number of altered samples in the whole dataset. C, Inference of CRISPR Edits (ICE) analyses of the edited region of the XRCC3 gene in WT (bottom) or XRCC3 KO (top) DU145 cells. ICE quantification reported an overall editing efficiency of 98% in the KO cells, with a 1 bp deletion accounting for 22% of the events and a 1 bp insertion accounting for the remaining 77%. Supplementary Figure S5. Half-maximal inhibitory concentration (IC50) for olaparib in the different isogenic cell lines used in this study as measured in clonogenic survival assays. Values were calculated from the dose-response curves in Figures 1, 2 and 4 and Supplementary Figure S3.

PARP inhibitors (PARPi) are currently indicated for the treatment of ovarian, breast, pancreatic, and prostate cancers harboring mutations in the tumor suppressor genes BRCA1 or BRCA2. In the case of ovarian and prostate cancers, their classification as homologous recombination repair (HRR) deficient (HRD) or mutated also makes PARPi an available treatment option beyond BRCA1 or BRCA2 mutational status. However, identification of the most relevant genetic alterations driving the HRD phenotype has proven difficult and recent data have shown that other genetic alterations not affecting HRR are also capable of driving PARPi responses. To gain insight into the genetics driving PARPi sensitivity, we performed CRISPR-Cas9 loss-of-function screens in six PARPi-insensitive cell lines and combined the output with published PARPi datasets from eight additional cell lines. Ensuing exploration of the data identified 110 genes whose inactivation is strongly linked to sensitivity to PARPi. Parallel cell line generation of isogenic gene knockouts in ovarian and prostate cancer cell lines identified that inactivation of core HRR factors is required for driving in vitro PARPi responses comparable with the ones observed for BRCA1 or BRCA2 mutations. Moreover, pan-cancer genetic, transcriptomic, and epigenetic data analyses of these 110 genes highlight the ones most frequently inactivated in tumors, making this study a valuable resource for prospective identification of potential PARPi-responsive patient populations. Importantly, our investigations uncover XRCC3 gene silencing as a potential new prognostic biomarker of PARPi sensitivity in prostate cancer.Significance:This study identifies tumor genetic backgrounds where to expand the use of PARPis beyond mutations in BRCA1 or BRCA2. This is achieved by combining the output of unbiased genome-wide loss-of-function CRISPR-Cas9 genetic screens with bioinformatics analysis of biallelic losses of the identified genes in public tumor datasets, unveiling loss of the DNA repair gene XRCC3 as a potential biomarker of PARPi sensitivity in prostate cancer.

CRISPR-Cas9 loss-of-function screens analysed in this study.

Supplementary Figure from Preclinical Characterization of AZD5305, A Next-Generation, Highly Selective PARP1 Inhibitor and Trapper

Supplementary Data from Preclinical Characterization of AZD5305, A Next-Generation, Highly Selective PARP1 Inhibitor and Trapper

Supplementary Figure S1. Analytical characterization of INT016-ADCs; Supplementary Figure S2. Validation of B7-H4 antibodies for use in IHC; Supplementary Figure S3. Relationship between B7-H4 expression level and heterogeneity in the studied human triple-negative cohort; Supplementary Figure S4. Binding of antibody INT016 to HEK 293 cells stably expressing human, mouse, or cynomolgus monkey B7-H4; Supplementary Figure S5. Binding of antibody INT016 to human breast cancer cell lines and to HT29 cells stably expressing human B7-H4; Supplementary Figure S6. Internalization and lysosomal trafficking of INT016; Supplementary Figure S7. In vitro stability of INT016-ADCs in mouse and cynomolgus monkey serum; Supplementary Figure S8. In vitro cytotoxicity of INT016-ADCs in HT29 and HT29-huB7-H4 isogenic cell lines; Supplementary Figure S9. In vivo efficacy of INT016-ADCs in the MX-1 xenograft model; Supplementary Figure S10. Growth rate analysis of MX-1 in vivo efficacy study comparing INT016-ADCs from days 14–40.

Surface plasmon resonance; In vitro cytotoxicity assays; In vitro proliferation assays; Internalization assays; Subcellular localization microscopy; Immunoblotting; Statistical analysis

Purpose:We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve antitumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to play a role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim of achieving improved clinical efficacy and wider therapeutic window. This next-generation PARP inhibitor (PARPi) could provide a paradigm shift in clinical outcomes achieved by first-generation PARPi, particularly in combination.Experimental Design:AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping, and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematologic toxicity was evaluated in rat models, as monotherapy and combination.Results:AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first-generation PARPi, AZD5305 has minimal effects on hematologic parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1 mg/kg once daily achieved greater depth of tumor regression compared to olaparib 100 mg/kg once daily, and longer duration of response.Conclusions:AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain the therapeutic benefit of nonselective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in phase I trials (NCT04644068).

Supplementary Table S1. Primary antibodies used for immunoblotting; Supplementary Table S2. Characterization of ADCs evaluated in this study; Supplementary Table S3. B7-H4 expression in human normal tissue; Supplementary Table S4. Binding affinity of anti-B7-H4 antibody INT016 Fab to human, cynomolgus monkey, and mouse B7-H4, measured by surface plasmon resonance.

Purpose:We evaluated the activity of AZD8205, a B7-H4–directed antibody–drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models.Experimental Design:IHC and deep-learning–based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly–Gly–Phe–Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models.Results:Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala–PEG8–TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker–payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4–expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance.Conclusions:These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4–expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482).See related commentary by Pommier and Thomas, p. 991

Supplementary Figures 1-4 from Inhibition of Histone Deacetylase in Cancer Cells Slows Down Replication Forks, Activates Dormant Origins, and Induces DNA Damage

Figure S1 shows absence of drug-related toxicity

Figure S1. Genomic analysis of SWI/SNF alterations in human cancer. Figure S2. SMARCA4-deficient lung cancer cells depend on SMARCA2 expression for growth. Figure S3. In-situ cell extraction assay for PFI-3 treatment. Figure S4. SMARCA2/4 bromodomain inhibitor does not alter growth of lung cancer cells. Figure S5. Analysis of SMARCA4 target gene expression and promoter occupancy following prolonged treatment with PFI-3. Figure S6. Pharmacological PFI-3 inhibitor studies in leukemia cells. Figure S7. Pharmacological EZH2 inhibitor studies. Figure S8. Genome-wide microarray analysis of SMARCA2 knockdown in A549 cells reconstituted with SMARCA4 cDNAs. Table S1: Survey of genomic lesions in SWI/SNF across different cancer types. Table S2: Sequence information of shRNA's and siRNA's used in the study. Table S3. BROMOScan (DiscoveRx) profiling of PFI-3 (at 2uM) against 32 bromodomains. Table S4. Definition of gene sets (i.e. SMARCA2 knockdown signatures) from SMARCA4 rescue experiments in A549 cells. Table S5. Summary of GSEA normalized enrichment scores (NES) and false discovery rate (FDR) for rescue experiment using ATP-Dead, BRD-Mut and vector control and compared to WT.

Protein acetylation is a reversible process regulated by histone deacetylases (HDAC) that is often altered in human cancers. Suberoylanilide hydroxamic acid (SAHA) is the first HDAC inhibitor to be approved for clinical use as an anticancer agent. Given that histone acetylation is a key determinant of chromatin structure, we investigated how SAHA may affect DNA replication and integrity to gain deeper insights into the basis for its anticancer activity. Nuclear replication factories were visualized with confocal immunofluorescence microscopy and single-replicon analyses were conducted by genome-wide molecular combing after pulse labeling with two thymidine analogues. We found that pharmacologic concentrations of SAHA induce replication-mediated DNA damage with activation of histone γH2AX. Single DNA molecule analyses indicated slowdown in replication speed along with activation of dormant replication origins in response to SAHA. Similar results were obtained using siRNA-mediated depletion of HDAC3 expression, implicating this HDAC member as a likely target in the SAHA response. Activation of dormant origins was confirmed by molecular analyses of the β-globin locus control region. Our findings demonstrate that SAHA produces profound alterations in DNA replication that cause DNA damage, establishing a critical link between robust chromatin acetylation and DNA replication in human cancer cells. Cancer Res; 70(11); 4470–80. ©2010 AACR.

Supplementary Methods, Figure Legends

PARP inhibitors (PARPi) have changed the management of patients with ovarian cancer and their effectiveness has been demonstrated especially in homologous recombination repair–deficient tumors. These first-generation drugs target PARP1, but also PARP2 and other family members potentially responsible for adverse effects that limit their therapeutic potential and restrict their use in combination with chemotherapeutic agents.We investigated ovarian cancer patient-derived xenografts (OC-PDXs) to assess whether malignant progression could be impaired by a novel inhibitor selective for PARP1 (AZD5305) and to assess the potential of its combination with carboplatin (CPT), the standard-of-care for patients with ovarian cancer.In BRCA-mutated OC-PDXs, AZD5305 achieved greater tumor regressions and longer duration of response as well as a superior impairment of visceral metastasis and improved survival benefit compared with the first-generation dual PARP1/2 inhibitors.The combination of AZD5305 plus CPT was more efficacious than single agents. Subcutaneously growing tumors experienced regression that persisted after therapy stopped. Combination efficacy was greater against tumors that did not respond well to platinum, even at a dose at which AZD5305 monotherapy was ineffective. The combination therapy impaired metastatic dissemination and significantly prolonged the lifespan of mice bearing OC-PDXs in their abdomen. This combination benefit was evident even when CPT was used at suboptimal doses, and was superior to full-dose platinum treatment.These preclinical studies demonstrate that the PARP1-selective inhibitor AZD5305 retains and improves the therapeutic benefit of the first-generation PARPi, providing an opportunity to maximize benefits for this class of anticancer agents.Significance:Selective PARP1i AZD5305 can exceed the efficacy of first-generation PARPi, which target both PARP1 and PARP2, and potentiates the efficacy of CPT when given in combination. AZD5305 alone or in combination with platinum delayed visceral metastasis, ultimately extending the lifespan of OC-PDX–bearing mice. These preclinical models mimic the progression of the disease occurring in patients after debulking surgery, and are translationally relevant.

Poly(ADP-ribose) polymerase (PARP) inhibitors have transformed the therapeutic landscape for advanced ovarian cancer and expanded treatment options for other tumor types, including breast, pancreas, and prostate cancer. Yet, despite the success of PARP inhibitors in our current therapeutic armamentarium, not all patients benefit because of primary resistance, whereas different acquired resistance mechanisms can lead to disease progression on therapy. In addition, the toxicity profile of PARP inhibitors, primarily myelosuppression, has led to adverse events in a proportion of patients as monotherapy, and has limited the use of PARP inhibitors for certain rational combination strategies, such as chemotherapy and targeted therapy regimens. Currently approved PARP inhibitors are essentially equipotent against PARP1 and PARP2 enzymes. In this review, we describe the development of next-generation PARP1-selective inhibitors that have entered phase I clinical trials. These inhibitors have demonstrated increased PARP1 inhibitory potency and exquisitely high PARP1 selectivity in preclinical studies-features that may lead to improved clinical efficacy and a wider therapeutic window. First-in-human clinical trials seeking to establish the safety, tolerability, and recommended phase II dose, as well as antitumor activity of these novel agents, have commenced. If successful, this next-generation of PARP1-selective agents promises to build on the succeses of current PARP inhibitor treatment paradigms in cancer medicine.

Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are currently indicated for the treatment of ovarian, breast, pancreatic and prostate cancers harbouring mutations in the tumour suppressor genes BRCA1 or BRCA2. In the case of ovarian and prostate cancers, their classification as homologous recombination repair (HRR) deficient (HRD) or mutated (HRRm) also makes PARPi an available treatment option beyond BRCA1 or BRCA2 mutational status. However, identification of the most relevant genetic alterations driving the HRD phenotype has proven difficult and recent data have shown that other genetic alterations not affecting HRR are also capable of driving PARPi responses. To gain insight into the genetics driving PARPi sensitivity, we performed CRISPR-Cas9 loss-of-function screens in 6 PARPi-insensitive cell lines and combined the output with published PARPi datasets from 8 additional cell lines. Ensuing exploration of the data identified 110 genes whose inactivation is strongly linked to sensitivity to PARPi. Parallel cell line generation of isogenic gene knockouts in ovarian and prostate cancer cell lines identified that inactivation of core HRR factors is required for driving in vitro PARPi responses comparable to the ones observed for BRCA1 or BRCA2 mutations. Moreover, pan-cancer genetic, transcriptomic and epigenetic data analyses of these 110 genes highlight the ones most frequently inactivated in tumours, making this study a valuable resource for prospective identification of potential PARPi-responsive patient populations. Importantly, our investigations uncover XRCC3 gene silencing as a potential new prognostic biomarker of PARPi sensitivity in prostate cancer.Statement of significanceThis study identifies tumour genetic backgrounds where to expand the use of PARP inhibitors beyond mutations in BRCA1 or BRCA2. This is achieved by combining the output of unbiased genome-wide loss-of-function CRISPR-Cas9 genetic screens with bioinformatics analysis of biallelic losses of the identified genes in public tumour datasets, unveiling loss of the DNA repair gene XRCC3 as a potential biomarker of PARP inhibitor sensitivity in prostate cancer.

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.

Purpose: We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve antitumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to play a role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim of achieving improved clinical efficacy and wider therapeutic window. This next-generation PARP inhibitor (PARPi) could provide a paradigm shift in clinical outcomes achieved by first-generation PARPi, particularly in combination. Experimental Design: AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping, and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematologic toxicity was evaluated in rat models, as monotherapy and combination. Results: AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first-generation PARPi, AZD5305 has minimal effects on hematologic parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1 mg/kg once daily achieved greater depth of tumor regression compared to olaparib 100 mg/kg once daily, and longer duration of response. Conclusions: AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain the therapeutic benefit of nonselective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in phase I trials (NCT04644068).

The cell-surface glycoprotein B7-H4 is overexpressed in a range of solid tumors including breast cancer, ovarian serous carcinoma, endometrial carcinoma, and cholangiocarcinoma, yet has limited expression in normal tissue, making it an attractive target for an antibody-drug conjugate (ADC). This presentation describes for the first time the development of AZD8205, a B7-H4 targeted ADC incorporating a novel topoisomerase 1 inhibitor (TOP1i) linker-warhead, AZ’0133 which was designed to exploit the full potential of B7-H4 as an ADC target. Initially, we investigated a series of more than 35 TOP1i compounds as warheads and achieved activity in a clinically relevant nM range. We further optimized the conjugation site and chemistry to reduce the potential for aggregation while maintaining potency, overcoming major synthetic challenges to deliver a robust synthetic route amenable to scale-up. Finally, with a series of optimized linker-warheads, we explored the impact of linker-warhead design on ADC hydrophobicity, stability, efficacy, pharmacokinetics and tolerability culminating in the development of AZD8205. The primary mechanism of action of AZD8205 is intracellular delivery of the TOP1i warhead to B7-H4 positive cells, leading to DNA damage and apoptotic cell death. AZD8205 drove bystander killing of target negative cells in mixed cultures in vitro, which is further supported by robust antitumor activity observed in in vivo studies with patient-derived xenograft (PDX) tumors with heterogeneous target expression, representing multiple tumor indications. In a study of 26 human TNBC PDX tumors, a single IV administration of 3.5 mg/kg AZD8205 provided an overall response rate of 69% (tumor regression of 30% or greater from baseline) and complete responses observed in 9/26 (36%) of models. To understand the biology underlying antitumor response, we conducted a multiparametric analysis including genomics, proteomics and computational pathology and found that deeper antitumor activity was observed in models with elevated B7-H4 expression as well as in models with defects in DNA damage repair (DDR). To further exploit the DNA damage elicited by the TOP1i warhead, we examined combinations of AZD8205 with small molecules, including a novel PARP1 selective inhibitor, in a BRCA wild type MDA-MB-468 model. These data suggest that AZD8205 is a promising therapeutic candidate for the treatment of B7-H4 positive solid tumors. A first in human phase 1 study in patients with advanced solid tumors is currently ongoing (NCT05123482). Citation Format: Krista Kinneer, Niall J. Dickinson, Luke Masterson, Thais Cailleau, Ian Hutchinson, Balakumar Vijayakrishnan, Nazzareno Dimasi, R. James Christie, Mary McFarlane, Kathryn Ball, Arthur Lewis, Sofia Koch, Lee Brown, Yue Huang, Anton I. Rosenbaum, Jiaqi Yuan, Si Mou, Noel R. Monks, Jon Chesebrough, Ravinder Tammali, Judith Anderton, Darrin Sabol, Frances Anne Tosto, Philipp Wortmann, Zachary A. Cooper, Pauline Ryan, John Hood, Carlos Fernandez Teruel, Carlos Serra Traynor, Andy Pike, Michael Davies, Elisabetta Leo, Kimberly Cook, Nadia Luheshi, Philip W. Howard, Puja Sapra. Discovery and first disclosure of AZD8205, a B7-H4-targeted antibody-drug conjugate utilizing a novel topoisomerase I linker-warhead [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 1765.

PARP inhibitors exploit defects in DNA repair pathways to selectively target cancerous cells via PARP1 catalytic inhibition and PARP1 trapping onto the DNA. All known clinical PARP1 inhibitors bind at the same site at the catalytic center of the enzyme. However, despite this resemblance they show immensely different outcomes in terms of response rate in the clinic due to their varying degree of PARP trapping ability. Moreover, the first-generation PARP inhibitors were not optimized for selectivity across the PARP family potentially driving undesirable side effects, including intestinal toxicity from tankyrase inhibition or hematological toxicity from PARP2 inhibition. There has been strong rationale for the use of PARP inhibitors in neuro-oncology. However, the first-generation PARP inhibitors have limited CNS distribution as these drugs were not designed for brain penetration. Recently AstraZeneca has reported the discovery of AZD5305, a next generation PARP1 selective inhibitor and PARP1-DNA trapper which was not designed with a CNS penetrant profile. Given the unmet need of a brain penetrant PARP1 inhibitor, we set out to identify a highly potent and selective PARP1 inhibitor and trapper with CNS profile. In our next generation PARP1 inhibitor, we sought to retain the profile of AZD5305 and lower the efflux for CNS penetration. Despite the challenge of narrow SAR, we successfully used the structure- and property-based design approach to identify a brain penetrant PARP1 inhibitor and PARP1-DNA trapper. We used multiple medicinal chemistry maneuvers such as masking the hydrogen bond donors and core modifications to lower the efflux in order to achieve brain penetration. Further optimization of the nicotinamide mimetic core for potency and metabolic stability led us to the discovery of AZD9574.AZD9574 shows improved selectivity for PARP1 over PARP2 vs AZD5305 and retains its excellent selectivity over other PARP family members. It has low efflux in Caco2, MDCK-MDR1, and MDCK-MDR1-BCRP permeability assays and it also showed CNS penetration in rat and cynomolgus monkey. AZD9574 has excellent secondary pharmacology and acceptable physicochemical properties and good PK in preclinical species.In vitro, AZD9574 selectively inhibits the growth of BRCAm cell lines. Importantly, AZD9574 showed efficacy in an intracranial BRCA1m MDA-MB-436 xenograft model at doses of 3, 10 and 30 mg/kg QD, significantly extending the survival of tumor-bearing mice compared to vehicle control arm.In summary, AZD9574 is a next generation selective PARP1 inhibitor and trapper with CNS penetration. This profile makes it an ideal candidate for treating CNS malignancies or brain metastases that have a dependence on PARP inhibition either as single agent or in combination with other therapies. Citation Format: Avipsa Ghosh, Sudhir M. Hande, Amber Balazs, Derek Barratt, Sabina Cosulich, Barry Davies, Sébastien Degorce, Kevin Embrey, Sonja Gill, Anders Gunnarsson, Giuditta Illuzzi, Peter Johnström, Jordan Lane, Carrie Larner, Rachel Lawrence, Elisabetta Leo, Andrew Madin, Elizabeth Martin, Lisa McWilliams, Lenka O’Connor, Mark O’Connor, Jonathan Orme, Fiona Pachl, Martin Packer, Andy Pike, Philip Rawlins, Marianne Schimpl, Magnus Schou, Anna Staniszewska, Wenzhan Yang, James Yates, Andrew Zhang, XiaoLa Zheng, Stephen Fawell, Petra Hamerlik, Jeffrey Johannes. Structure-based and property-based drug design of AZD9574, a CNS penetrant PARP1 selective inhibitor and trapper [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 6302.

Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor. T-DXd is approved for patients with HER2+ metastatic breast cancer who have received two or more anti-HER2 based regimens as well as HER2+ gastric cancer, and has demonstrated antitumor activity in both HER2+ and HER2-low cancers clinically. Trapping of Topo1cc by DXd in targeted cells, induces replication stress (RS) and double strand breaks (DSB), activating a DNA Damage Repair (DDR) response. Therefore we aimed to investigate if combination of T-DXd with DDR inhibitors enhances efficacy and durability of response. Methods: We evaluated the antiproliferative effect of the combination of T-DXd with AZD6738 (ATR inhibitor), AZD1775 (WEE1 inhibitor), AZD1390 (ATM inhibitor), in a panel of 27 breast cancer cell lines in an in vitro 7-day viability assay. Combinations were also evaluated in vivo in the HER2-high gastric NCI-N87 cell line xenograft. To evaluate the specificity of the combination activity in tumor cells (vs normal tissue), we further evaluated the combination in a 2D in vitro human bone marrow progenitor assay. Results: We found that the combination with all DDRi significantly enhanced in vitro cell killing activity over single agents in many of the models tested (12/27 for AZD6738, 9/27 for AZD1775, 6/27 for AZD1390). This was not limited to models with DNA damage repair mutations (e.g. BRCA1, BRCA2, or ATM). Mechanistically, T-DXd activated PARP and the ATR, Wee1, ATM pathways, inducing cell cycle arrest. Combination of T-DXd with AZD6738 and AZD1775 inhibited the RS response (pATR-T1989, pChk1-S345) and cell cycle arrest (pCdc2-Y15), while AZD1390 inhibited the DSB response pathway (pATM-S1981, pRad50-S685, pKap1-S824, pChk2-T68). All DDR inhibitors exacerbated DNA damage (pRPA-S4/8, gH2AX) and cell death (cCasp3). In the in vitro human bone marrow (BM) assay, T-DXd had no synergistic interaction with AZD6738 and AZD1775 inhibitors, but synergy with AZD1390. In vivo, combination therapy with all DDRi tested achieved superior Tumor Growth Inhibition (TGI) compared to monotherapy alone. At day 41 in NCI-N87, 3mg/kg of T-DXd provided TGI of 74% while monotherapy arms of 25mg/kg BID AZD6738, 60mg/kg (5-days on/2-days off) AZD1775 or 10mg/kg AZD1390 provided 68%, 47%, 31% TGI respectively. The combinations of T-DXd with AZD6738, AZD1775 and AZD1390 provided TGI of >100% (19% regression), 90%, and 99% respectively. Conclusions: These results suggest that AZD6738, AZD1775, and AZD1390 all potentiate T-DXd activity in vitro and in vivo, and the combination interactions with AZD6738 and AZD1775 were much stronger in some tumor models over bone marrow cells in vitro. These data suggest further investigation of these combinations in clinical studies may be warranted. Citation Format: Yann Wallez, Theresa Proia, Azadeh Cheraghchi-Bashi-Astaneh, Ankur Karmokar, Zena Wilson, Suzanne Randle, Mark Anderton, Stephen Durant, Elisabetta Leo, Alan Lau, Mark O'Connor, Jerome Mettetal. Activity and tolerability of combinations of trastuzumab deruxtecan (T-DXd) with inhibitors of the DNA damage response in preclinical models [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 5298.

Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor, approved for HER2+ metastatic breast cancer. Clinically, T-DXd has demonstrated antitumor activity in both HER2+ and HER2-low cancers. Due to the role of PARP1 in resolution of DNA damage induced by topoisomerase I trapping, we tested the combination of the next generation PARP1-selective inhibitor AZD5305 with T-DXd. Methods: We evaluated the antiproliferative ability of the combination of T-DXd with AZD5305 in a panel of 27 breast cancer cell lines in an in vitro 7-day viability assay. The combination was also evaluated in vivo in two non-HRD HER2+ models, KPL4 (Breast) and NCI-N87 (Gastric) at doses of 3mg/kg and 10mg/kg Q3W for T-DXd combined with 0.01, 0.1, and 1 mg/kg QD of AZD5305. To evaluate the specificity of the combination activity in tumor cells (vs normal tissue), we further evaluated the combination in a human 2D in vitro bone marrow progenitor assay. Results: We found that the combination had enhanced in vitro cell killing activity over single agents in 8/27 of the models tested. The benefit was present in both Homologous Recombination Deficient (HRD) as well as Homologous Recombination proficient, suggesting it does not depend on HRD (as defined by mutations in DNA damage repair genes). Mechanistically, T-DXd activated PARP and the combination of T-DXd with AZD5305 abrogated PARP1 auto-parylation, leading to enhanced DNA damage (gH2AX, pRPA-S4/8) and cell death (cCasp3). In vivo, the combination was well tolerated and more active than monotherapy of either compound in both KPL4 (at 30 days the growth inhibition was 95% at 10mg/kg T-DXd, 10% at 1mg/kg AZD5305, and 100% TGI with 97% regression with T-DXd + AZD5305) and NCI-N87 (at 41 days TGI of 74% with 10mg/kg T-DXd, 47% with 1mg/kg AZD5305, and 100% TGI with 40% regression for T-DXd + AZD5305; p100% TGI with 72% regression on day 30) vs. monotherapy T-DXd alone (95% TGI). Conclusions: These results suggest that T-DXd combined with the next generation PARP1 inhibitor AZD5305 is a potentially active combination, with preclinical activity demonstrated in HRD and HR proficient models. Further, the dose and scheduling may warrant exploration clinically to optimize therapeutic index. Citation Format: Yann Wallez, Theresa Proia, Elisabetta Leo, Laura Bradshaw, Zena Wilson, Joe’l Owusu, Azadeh Cheraghchi-Bashi-Astaneh, Anna Staniszewska, Mark O’Connor, Sabina Cosulich, Jerome Mettetal. Activity and tolerability of combination of trastuzumab deruxtecan with the next generation PARP1-selective inhibitor AZD5305 in preclinical models [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 1142.

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.

Large independent analyses on cancer cell lines followed by functional studies have identified Schlafen 11 (SLFN11), a putative DNA/RNA helicase, as the strongest predictor of sensitivity to DNA-damaging agents. 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 specimens prior platinum-based chemotherapy treatment, we demonstrate that SLFN11 is expressed by infiltrating innate and adaptive immune cells. We show, for the first time, that SLFN11 density in both the neoplastic and microenvironmental components was independently associated with favorable outcome. Transcriptomic analyses suggested the presence of a hitherto modulation of the cancer-immunity cycle orchestrated by SLFN11. We propose SLFN11 as a dual biomarker capturing simultaneously interconnected immunological and cancercell-intrinsic functional dispositions associated with sensitivity to DNA damaging agents.

High grade serous ovarian cancer (HGSOC) is a major cause of female cancer mortality. The approval of poly (ADP-ribose) polymerase (PARP) inhibitors for clinical use has greatly improved treatment options for patients with homologous recombination repair (HRR)-deficient HGSOC, although the development of PARP inhibitor resistance in some patients is revealing limitations to outcome. A proportion of patients with HRR-proficient cancers also benefit from PARP inhibitor therapy. Our aim is to compare mechanisms of resistance to the PARP inhibitor olaparib in these two main molecular categories of HGSOC and investigate a way to overcome resistance that we considered particularly suited to a cancer like HGSOC, where there is a very high incidence of TP53 gene mutation, making HGSOC cells heavily reliant on the G2 checkpoint for repair of DNA damage and survival. We identified alterations in multiple factors involved in resistance to PARP inhibition in both HRR-proficient and -deficient cancers. The most frequent change was a major reduction in levels of poly (ADP-ribose) glycohydrolase (PARG), which would be expected to preserve a residual PARP1-initiated DNA damage response to DNA single-strand breaks. Other changes seen would be expected to boost levels of HRR of DNA double-strand breaks. Growth of all olaparib-resistant clones isolated could be controlled by WEE1 kinase inhibitor AZD1775, which inactivates the G2 checkpoint. Our work suggests that use of the WEE1 kinase inhibitor could be a realistic therapeutic option for patients that develop resistance to olaparib.

Since the approval of poly (ADP-ribose) polymerase inhibitors (PARPi) for BRCA-mutated ovarian cancer patients, different PARPi have been developed and have shown efficacy in homologous recombination deficient (HRD) tumours. These first-generation drugs inhibit both PARP1 and PARP2 (as well as other PARP family members) and present undesirable adverse effects, such as haematological and intestinal toxicity, that restricted their use especially in combination with already poorly tolerated chemotherapeutic agents. Here, patient-derived ovarian cancer xenografts (OC-PDXs) were used i) to evaluate the dose response efficacy of AZD5305, a next generation, potent and selective PARP1 inhibitor and ii) to investigate the effect of the combination with carboplatin, a standard-of-care treatment for ovarian cancer patients. AZD5305 was administered orally (po) once daily (QD) for 8 weeks as monotherapy (0.1, 1 and 10 mg/kg) and in combination with carboplatin (CPT) dosed intravenously (35 mg/kg; Q7x4). Two BRCA1 mutated (HOC106 and HOC107) and a BRCA wild-type (HOC84) OC-PDXs (all TP53 mutated) were implanted subcutis and the effect of treatments on tumour growth evaluated. AZD5305 dosed at 0.1, 1 and 10 mg/kg to mice bearing HOC106 tumours, inhibited tumours growth in a dose dependent manner. Administered in combination at 0.1 and 1 mg/kg, AZD5305 potentiated the effect of carboplatin, causing a significant and sustained regression of HOC106 tumours. The anti-tumour efficacy was also evident against the PARPi-resistant OC-PDX HOC107, whereby the tumour growth was inhibited by AZD5305 at 1 and 10 mg/kg dose. Combined with carboplatin, AZD5305 stabilized the growth of HOC107 tumours at doses as low as 0.1 mg/kg that on its own was not effective. As expected, none of the treatments affected the growth of the BRCA wild-type HOC84 (PARPi and carboplatin resistant OC-PDX). Overall, AZD5305 a next generation PARP1-selective inhibitor and trapper, shows improved efficacy and tolerability in combination with chemotherapy compared to first generation, PARP1/2 inhibitors, making it a promising clinical candidate for the treatment of ovarian cancer. Citation Format: Giulia Dellavedova, Alessandra Decio, Anna Staniszewska, Elisabetta Leo, Raffaella Giavazzi, Maria Rosa Bani. The next generation PARP inhibitor AZD5305 is active in a broad range of pre-clinical models of ovarian cancer [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 P217.

Since the approval of olaparib in 2014 for BRCA mutated (BRCAm) ovarian cancer, many PARP inhibitors have been developed and have seen widespread success. However, as a class, these drugs are not without adverse events which have limited their ability to be combined with chemotherapy. Most first generation PARP inhibitors were developed and optimized before the concept of PARP1-DNA trapping was discovered as the mechanism by which PARP inhibitors exert their synthetic lethal effects on BRCAm cells. Moreover, the first generation PARP inhibitors were not optimized for selectivity across the PARP family potentially driving undesirable side effects, including intestinal toxicity from tankyrase inhibition or hematological toxicity from PARP2 inhibition. With this in mind, we set out to discover a best-in-class, second generation PARP inhibitor that was highly selective for PARP1 over the other 16 members of the PARP family, as well as a highly potent PARP1-DNA trapper. PARP1 and PARP2 have a highly similar amino acid sequence, and most of the residues around the nicotinamide binding site are identical. However, there are some key residue differences in the helical domain which serves a regulator of the nicotinamide binding pocket. The publication of NMS-P118 in 2015 by Nerviano Medical Sciences showed that a highly selective PARP1 inhibitor could be found. This work inspired us to screen an extensive list of previously reported PARP inhibitors for selectivity against PARP2 and we found that FR257516 met the selectivity criteria as previously reported, but lacked the ability to trap PARP1 to DNA and hence lacked any activity in a cell colony formation assay in DLD-1 BRCA2-/- cells. Using parallel chemistry to generate diverse analogs, X-ray crystallography to enable structure-based design, and exploration of multiple nicotinamide mimetic cores, we were able to generate lead compound AZ4554, which was a PARP1 selective PARP1-DNA trapper with potent activity in BRCAm cells. Using concepts of property-based drug design, we were able to optimize lead compound AZ4554 into candidate drug AZD5305, making key improvements in secondary pharmacology, including reducing hERG activity, and intrinsic clearance in human microsomes through the introduction of polar atoms to lower logD without compromising permeability or oral bioavailability. AZD5305 is a highly selective binder of PARP1 over PARP2 and other PARP enzymes by fluorescence polarization, surface plasmon resonance, and single molecule spectroscopy. It is highly potent against DLD-1 BRCA2-/- cells, while sparing isogenic BRCA WT cells. The secondary pharmacology of AZD5305 is remarkably clean, with hERG activity >40 µM. AZD5305 has a very favorable pre-clinical PK profile, low predicted human dose, and has shown efficacy in an MDA-MB-436 mouse xenograft model. Citation Format: Sudhir Hande, Amber Balazs, Sébastien L. Degorce, Kevin Embrey, Avipsa Ghosh, Sonja J. Gill, Anders Gunnarsson, Giuditta Illuzzi, Jordan Lane, Carrie Larner, Elisabetta Leo, Andrew Madin, Lisa McWilliams, Mark J. O'Connor, Jonathan Orme, Fiona Pachl, Martin Packer, Andy Pike, Philip Rawlins, Marianne Schimpl, Anna D. Staniszewska, Andrew Zhang, Xiaolan Zheng, Jeffrey W. Johannes. Structure-based and property-based drug design of AZD5305, a highly selective PARP1 inhibitor and trapper [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 296.

Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown efficacy in homologous recombination deficient (HRD) tumours, such as those with BRCA mutations (BRCAm). In this setting PARPi treatments lead to accumulation of DNA damage and cancer cell death. PARPi currently in clinical use inhibit both PARP1 and PARP2, as well as other members of the PARP family. Here, we report for the first time in vivo profiling of AZD5305, a potent and highly selective PARP1 inhibitor and trapper, currently in Ph1 clinical trials. Dose response efficacy of AZD5305 was evaluated in the BRCA1m triple-negative breast cancer (TNBC) xenograft model MDA-MB-436. AZD5305 dosed at 0.1mg/kg QD or higher for 35 days delivered about 90% regression, compared with 83% regression caused by treatment with 100mg/kg QD olaparib. Anti-tumour effects of AZD5305 continued after cessation of treatment and complete responses were achieved which were sustained for the whole duration of the study, over 100 days after treatment withdrawal, in contrast to the olaparib-treated group where regrowth of tumours was observed from day 63 after treatment withdrawal. Investigation of the PK/PD/efficacy relationship in MDA-MB-436 showed that maximum efficacy of AZD5305 was achieved when unbound plasma concentrations were maintained above the IC95 estimated from an in vitro DLD-1 BRCA2-/- cell growth assay. Similar results were obtained in a BRCA1m patient-derived explant (PDX) model, HBCx-17. Anti-tumour efficacy of AZD5305 was also tested in the DLD-1 BRCA2-/- and wild-type (WT) isogenic xenograft models. In the DLD-1 BRCA2-/- model, AZD5305 dosed at 10mg/kg QD and 1mg/kg QD delivered 78% and 63% tumour regression, respectively. AZD5305 at 0.1mg/kg QD resulted in responses similar to those observed in the olaparib 100mg/kg QD group (40-54% tumour growth inhibition, TGI). As expected, AZD5305 and olaparib showed no anti-tumour efficacy in the DLD-1 WT tumour model. Due to improved PARP1 selectivity, AZD5305 has the potential to show improved efficacy and tolerability in combination with standard of care chemotherapy when compared to non-selective PARPi. Hence, we investigated the anti-tumour effects of AZD5305 in combination with carboplatin or paclitaxel in a BRCA1m TNBC xenograft, SUM149PT, and BRCA WT TNBC PDX model, HBCx-9. In both models, combination of AZD5305 with carboplatin was well tolerated and demonstrated clear benefit compared to each monotherapy treatment. The effects of adjusted dosing and scheduling of the combination on the anti-tumour efficacy will be presented. Citation Format: Anna D. Staniszewska, James W. Yates JWT, Andy Pike, Christine Fazenbaker, Kimberly Cook, Emily Bosco, Aaron Smith, Joanne Wilson, Elisabetta Leo. The novel PARP1-selective inhibitor, AZD5305, is efficacious as monotherapy and in combination with standard of care chemotherapy in the in vivo preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1270.

Poly ADP-ribose polymerase (PARP) inhibitors have demonstrated clinical effectiveness in the treatment of homologous recombination deficient tumors; however, hematological and other toxicities have limited their use as a class of agents, both as monotherapy and as combination with already poorly tolerated chemotherapies. Recent literature suggests that inhibition of PARP2, a feature shared by all the current clinical PARP inhibitors, is not required for efficacy and may in fact be associated with hematological effects. These observations led to our hypothesis that a highly selective and potent PARP1 inhibitor and trapper would retain efficacy while improving therapeutic index, thus expanding clinical combinations options. A high throughput screen against PARP1 and PARP2 identified a compound series with selectivity for binding to PARP1 over PARP2. This set of compounds was refined further by screening for the ability to trap PARP1 to DNA and selectively inhibit growth of BRCA mutant cell lines. Further lead optimization efforts utilizing PARP1 enzyme structural information, lead to the discovery of to AZD5305. AZD5305 is a highly potent inhibitor of PARP1, with significant PARP1-DNA trapping activity, no PARP2-activity, nor binding activity to any other members of the PARP family. AZD5305 has excellent secondary pharmacology and physicochemical properties as well as high oral bioavailability in preclinical species. AZD5305 has shown unique properties in preclinical toxicology models. When dosed daily for 14 days in rats at clinically equivalent doses, olaparib (a PARP1/2 inhibitor), causes up to 50% reduction in hemoglobin, in line with clinically observed anemia. Under the same experimental conditions, AZD5305 had no effects on any hematological parameters measured, when dosed at predicted clinically efficacious doses. In vitro, AZD5305 inhibits growth selectively in cell lines with deficiencies in DNA repair pathways, with IC50s in the single-digit nM range, while having no or minimal growth inhibitory effects in other cells (IC50s >10μM). The difference observed between the effects on DNA repair deficient and proficient cell lines is unprecedented with PARP inhibitors and indicates the great potential for AZD5305 to demonstrate an improved therapeutic index in the clinic. Treatments of xenografts and PDX models with AZD5305 at doses ≥ 0.1 mg/kg QD, compared to olaparib 100 mg/kg QD, resulted in greater depth of tumor regressions, as well as significantly longer duration of regressions following cessation of dosing. Overall, AZD5305 is a next generation PARP inhibitor and trapper, with enhanced anticancer efficacy and an improved safety profile in preclinical models compared to other PARP inhibitors. These features make AZD5305 an exciting and unique clinical candidate, with multiple clinical options as monotherapy and in combination. AZD5305 is in Ph1 clinical trials. Citation Format: Elisabetta Leo, Jeffrey Johannes. Discovery and first structural disclosure of AZD5305: A next generation, highly selective PARP1 inhibitor and trapper [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr ND05.

PARP inhibitor treatments are synthetically lethal with BRCA1/2 mutations and, in this setting, accumulation of DNA damage leads ultimately to cell death. Increases in DNA damage are also associated with increased levels of cytosolic DNA and consequently signalling through the cGAS-STING pathway, which can lead to increased inflammatory gene expression and Type I interferon response. We sought to evaluate the benefit of combining the PARP inhibitor olaparib with immune checkpoint blockade (ICB) in a mouse syngeneic ovarian model, BR5, which lacks BRCA1. We show that olaparib combination with anti-PD-L1 prolonged duration of anti-tumor response vs. either monotherapy. The combination increased durable complete responses (CRs) with 6/10 animals tumor free at 77 days, compared with 1/9 and 2/9 tumor-free animals for olaparib and anti-PD-L1 monotherapies, respectively. Similar results were observed for combination of olaparib with anti-CTLA-4. Furthermore, mice treated with olaparib + ICB demonstrated immunological memory, with 100% of mice with CRs successfully rejecting tumor growth upon rechallenge with BR5. Olaparib treatment of human T cells in vitro and mouse T cells in vitro and in vivo did not show inhibition of T cell activation or proliferation. Moreover, olaparib treatment led to changes in tumor immune infiltrate in the BR5 tumors, including approximately 2-fold increased CD8 T cells and 1.5-fold increased NK cells relative to vehicle. Differential gene expression analysis of olaparib or anti-PD-L1treated BR5 tumors revealed broad upregulation of immune pathways. Interestingly, type I IFN and STING pathways showed a more pronounced upregulation with Olaparib than with anti-PD-L1 treatment. Moreover, a dose-dependent upregulation of immune pathways, including JAK-STAT, STING and type I IFN was observed in a tumor cell-centric analysis from a BRCA1mut breast PDX model treated with olaparib. In contrast, no significant upregulation of STING or type I IFN pathways is observed in response to olaparib in a BRCAwt breast PDX model. In vitro mechanistic studies demonstrated that co-culture of olaparib-treated human BRCA1mut MDA-MB-436 tumor cells with human dendritic cells (DC) resulted in an approximately 2-fold upregulation of CD86 expression on DCs. Comparison of isogenic DLD-1 tumor cells showed CD86 upregulation on DCs only following co-culture with olaparib-treated BRCA2-/- DLD-1 cells, but not wt DLD-1 cells. Similarly, increased PD-L1 expression was only observed in BRCA2-/- DLD-1 cells treated with olaparib and following co-culture with DC. These observations suggest a basis for increased immune priming following olaparib treatment of BRCAmut tumors, which can be consolidated with ICB, such as anti-PD-L1, treatment. Citation Format: Anna Staniszewska, Joshua Armenia, Matthew King, Chrysiis Michaloglou, Maneesh Singh, Maryann San Martin, Zena Wilson, Theresa Proia, Oona Delpuech, Mark O'Connor, Elisabetta Leo, Viia Valge-Archer. Anti-tumor and immune effects of olaparib +/- anti-PD-L1 in preclinical BRCA1mut tumor models [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 967.

The DNA damage response (DDR) safeguards genome stability and promotes cellular survival by counteracting deleterious consequences of DNA damage. Deficiencies in DDR pathways contribute to genome instability, a hallmark of cancer development, and targeting the DDR is a promising anti-cancer therapy strategy. We describe here in vitro and in vivo studies allowing mechanistic understanding of the benefit of combining our DDR kinase inhibitors (DDRi) targeting ATM (AZD0156), ATR (AZD6738) and DNA-PK (AZD7648) with the PARP inhibitor, olaparib, in homologous recombination-deficient backgrounds. Using the break-induced replication (BIR) assay, which measures homologous-recombination repair (HRR) of DNA double-strand breaks resembling broken replication forks, we observed that ATRi decreases this type of homologous recombination repair by up to 50%, while inhibiting DNA-PK or ATM showed no effect. This provides a mechanistic rationale for combining ATRi with compounds that cause replication-fork collapse, such as PARPi. Indeed, combination of olaparib with the ATRi in BRCA1 mutant UWB1.289 cells enhanced olaparib sensitivity a thousand-fold (GI50 from 590 nM to 0.6 nM). This correlates with the complete tumour growth inhibitions (TGI) observed for the ATRi + olaparib combination in the BRCA2 mutant HBCx-10 triple negative breast cancer PDX model and is consistent with a synergistic increase of genome instability measured by metaphase spread analysis (total aberrations: olaparib; 11, ATRi; 8, combination; 35). DNA-PKi, however, did not synergise with olaparib, showing little enhancement of olaparib single-agent treatment (GI50 from 0.59 μM to 0.33 μM) and no significant impact on the total amount of chromosomal aberrations (total aberrations: olaparib; 11, DNA-PKi ; 3, combination; 14), suggesting that DNA-PKi alone or in combination with olaparib have little impact on BRCA1 mutated tumours. In ATM KO FaDu xenografts, ATRi or DNA-PKi single-agent treatment resulted in TGI, with complete tumour regressions when combined. Focusing on the olaparib combination, metaphase spread analysis of ATRi-treated FaDu ATM KO cells showed an enhancement of DNA replication-dependent chromatid breaks (1.3 vs 0.2 breaks/spread in ATM WT), while DNA-PKi treatment predominantly caused chromosome breaks (1.7 vs 0.2 breaks/spread in ATM WT) that are not dependent on replication. When combined with olaparib, ATRi acted synergistically and in a manner similar to that observed in the BRCA1 mutant cells, while the DNA-PKi + olaparib combination was additive. These data suggest that enhanced olaparib sensitisation of ATM KO cells by ATRi or DNA-PKi combinations occurs through different mechanisms. Together, these findings provide mechanistic differentiation of our DDRi in specific genetic backgrounds. This work informs how to clinically position these agents to benefit the right patients not only as single-agent treatments, but also in combination. Citation Format: Josep V. Forment, Paul Wijnhoven, Antonio Ramos-Montoya, Jacqueline Fok, Valeria Follia, Mercedes Vazquez-Chantada, Rajesh Oderda, Zena Wilson, Alan Lau, Elisabetta Leo, Stephen Durant, Elaine Cadogan, Mark J. O'Connor. Mechanistic differentiation of targeted DDR agent combinations [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 626.

AstraZeneca obtained positive results from POLO, the first Phase III randomised, double-blind study that evaluated the efficacy of the PARP inhibitor (PARPi) olaparib, for maintenance monotherapy in patients with gBRCA mutated metastatic pancreatic cancer whose disease has not progressed on first-line platinum-based chemotherapy. To support the application of olaparib for the proposed indication in these patients, a series of in vitro pharmacological tests were performed and will be here presented. 1. In a panel of pancreatic cancer cell lines, a striking correlation was observed between the responses to platinum and to olaparib. This indicates that it is predictable that olaparib will show efficacy in treatment of platinum-sensitive pancreatic cancer, as it has been previously demonstrated in other cancer indications. 2. Tests in isogenic cell line pairs and non-tumour cell lines revealed that platinum treatments lead to cytotoxic effects that are only ~10-20-fold more selective for BRCAm versus BRCAwt and non-tumour models; in contrast, olaparib is able to selectively kill BRCAm cells, with ~500-1000-fold sensitivity versus the BRCAwt cells. 3. In the panel of pancreatic cancer cells, the sensitivity to other DNA damaging agents used as standard of care, such as irinotecan (and, to some extent, 5FU, other key components of FOLFIRINOX as well as gemcitabine), correlated with sensitivity to olaparib. In contrast, no correlation was observed with paclitaxel (mitotic inhibitor), or with other agents. 4. The antiproliferative effects to olaparib treatments in the cancer cell panel were compared to that of other 5 clinical PARPi. High correlations were observed in the responses to all the PARPi except for veliparib, which inhibits the catalytic activity of PARP but does not act as PARP-trapper, confirming that the efficacy of PARPi in monotherapy depends on their ability to stabilise the interaction between PARP and the DNA. Citation Format: Elisabetta Leo, Giuditta Illuzzi, Sabrina Bentouati. Olaparib antiproliferative effect in pancreatic cancer and correlation with the response to other anticancer 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 1829.

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.

Nowadays growth of on board driver assistance systems is transforming the vehicles into systems that continuously and always more need to be fed by reliable and safe environmental information. A key-role information is represented by the road surface condition. A new digital application has been designed in order to allow all the personal devices (smartphones, smartwatches and wearables) to collaborate, recording kinematic quantities and sending the data to a target server. The central artificial intelligence collects all the receiving data populating a geo-database. The logic of identification estimate the so called Most Probable Friction (MPF), both time and position dependent. With scheduled periodicity, the cloud sends the refreshed MPF to all the connected users aimed to improve driving safety.

SIRT1, the mammalian homolog of yeast Sir2, is a founding member of a family of 7 protein and histone deacetylases that are involved in numerous biological functions. Previous studies revealed that SIRT1 deficiency results in genome instability, which eventually leads to cancer formation, yet the underlying mechanism is unclear. To investigate this, we conducted a proteomics study and found that SIRT1 interacted with many proteins involved in replication fork protection and origin firing. We demonstrated that loss of SIRT1 resulted in increased replication origin firing, asymmetric fork progression, defective intra-S-phase checkpoint, and chromosome damage. Mechanistically, SIRT1 deacetylates and affects the activity of TopBP1, which plays an essential role in DNA replication fork protection and replication origin firing. Our study demonstrated that ectopic over-expression of the deacetylated form of TopBP1 in SIRT1 mutant cells repressed replication origin firing, while the acetylated form of TopBP1 lost this function. Thus, SIRT1 acts upstream of TopBP1 and plays an essential role in maintaining genome stability by modulating DNA replication fork initiation and the intra-S-phase cell cycle checkpoint.

Treatments with PARPi in cancers with impaired DNA repair mechanisms (i.e. with Homologous Recombination Repair Deficiency, HRD) causes unsupportable genomic instability resulting in tumor cell death. PARPi act via a dual mechanism: 1) they block PARylation activity that normally occurs in response to DNA damage; 2) they trap PARP onto DNA lesions creating potentially cytotoxic PARP-DNA complexes. The longer PARP is inhibited and trapped onto the DNA, the greater the cytotoxic effect of PARPi in preclinical models. However, differences in preclinical PARP trapping potency have not translated into increased clinical efficacy with standard clinical doses used. Therefore, the ability to assess PARP-chromatin trapping in cancer models is critical for understanding the MoA of existing clinical PARPi. To date, this has been achieved by low throughput assays in non-HRD models using high PARPi concentrations and the addition of exogenous DNA damage. Here, we describe the development of a novel assay, a high throughput in situ cell extraction platform, where PARP-chromatin trapping is monitored by immunofluorescence and can be multiplexed with the analysis of other relevant biomarkers. Our novel assay has several advantages compared to conventional methods: the overall experimental process is simpler and less time consuming with results being quantitative and less error prone. Most importantly, the higher throughput allows a thorough evaluation of PARP1-chromatin trapping kinetics and their effects with dose-response in a time-dependent manner for clinical PARPi following both continuous treatment or after a wash out of drug. Using this new assay, we tested the kinetics in isogenic cell line pairs (BRCA2-/- and WT) and followed in parallel the appearance of biomarkers of DNA damage (e.g. γ-H2AX). The data have revealed important elements of differentiation between the MoA of clinical PARPi including the important insight that the strongest PARP trapper (talazoparib) has clearly reduced cytotoxic specificity for HRD cells, which likely explains the significantly reduced dose used in the clinic compared to other PARPi. These along with other data presented highlight how this next generation PARP trapping assays can provide important insights into PARPi MoA. Citation Format: Giuditta Illuzzi, Mark J O'Connor, Elisabetta Leo. A novel assay for PARP-DNA trapping provides insights into the mechanism of action (MoA) of clinical PARP inhibitors (PARPi) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2077.

Introduction: SCLC is an aggressive high-grade neuroendocrine malignancy in which targeting DNA-damage repair pathways has shown efficacy in pre-clinical and clinical contexts. We previously conducted a phase I/II trial combining the PARP inhibitor olaparib (O) tablets with the alkylating agent temozolomide (T) in patients with relapsed SCLC, in which O and T were both given days (d) 1-7 of each 21 d cycle. The recommended phase 2 dose (RP2D) was O 200 mg PO BID and T 75 mg/m2 PO daily, and the response rate among the first 29 evaluable patients was 41.4%, with the primary toxicities being hematologic (Farago et al., ASCO 2018). Continuous PARP inhibition with O may improve efficacy, though it is unknown how best to incorporate T onto this backbone. Simultaneous clinical testing of multiple dosing schedules for concurrent OT would be impractical. Here we present an optimization of the OT regimen using PDX models generated from patients treated with OT. Methods: SCLC PDX models derived from two patients prior to their durable responses to OT, MGH1518-1B and MGH1528-1, were used for dose optimization. Based on a human-murine comparison of serum Cmax for OT, the estimated murine-equivalents for the RP2D doses were O 25 mg/kg BID and T 6.25 mg/kg/d. The RP2D regimen was then modulated in both dose intensity and duration in a 9-arm PDX trial. The arms included 3 schedule categories, with doses varied within each category: discontinuous regimens (OT d 1-7); continuous O regimens (O d1-21 + T d 1-7); and rapid- or slow-alternating regimens administering O and T on non-overlapping days. Tumor bearing mice were treated when subcutaneous tumors reached a volume of 400-800 cc, enabling measurement of tumor regression (PDX response) and time to volume doubling (PDX TTP). Findings: The RP2D regimen resulted in near-complete response in both models. This degree of regression was recapitulated with nearly all dosing schedules, and was not improved by increasing T to 2x-RP2D. However, the PDX TTP varied. In both models, the longest TTP was seen with continuous O at full- or half-RP2D and d 1-7 T at half-RP2D. This regimen prolonged TTP by 12% in MGH1518-1B and 20% in MGH1528-1, versus discontinuous OT at RP2D. Based on these results, we initiated clinical treatment in a new cohort of patients on OT, with a phase 1 dose escalation starting with O 50 mg BID d 1-21 and T 50 mg/m2 d 1-7 of each 21 d cycle (NCT02446704). At this first dose level, a partial response by RECIST 1.1 criteria was observed in a heavily pre-treated patient, indicating clinical activity of this combination with this new dosing strategy. Conclusions: In PDX models sensitive to OT at our previously determined RP2D, we find that continuous O with intermittent T permits dose reduction of both O and T without loss of efficacy. A clinical trial is ongoing to determine whether this strategy may enable longer-term dosing and improve efficacy in patients. Citation Format: Benjamin J. Drapkin, Sarah Phat, David T. Myers, Jun Zhong, Beow Y. Yeap, Elisabetta Leo, Anna Stansiszewska, Aaron Smith, Elaine Cadogan, Maria C. Pietanza, Nicholas J. Dyson, Anna Farago. Dose optimization of olaparib plus temozolomide in small cell lung cancer (SCLC) patient-derived xenograft (PDX) models for clinical translation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4736.

The eukaryotic DNA replication initiation factor Mcm10 is essential for both replisome assembly and function. Human Mcm10 has two DNA-binding domains, the conserved internal domain (ID) and the C-terminal domain (CTD), which is specific to metazoans. SIRT1 is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that belongs to the sirtuin family. It is conserved from yeast to human and participates in cellular controls of metabolism, longevity, gene expression and genomic stability. Here we report that human Mcm10 is an acetylated protein regulated by SIRT1, which binds and deacetylates Mcm10 both in vivo and in vitro, and modulates Mcm10 stability and ability to bind DNA. Mcm10 and SIRT1 appear to act synergistically for DNA replication fork initiation. Furthermore, we show that the two DNA-binding domains of Mcm10 are modulated in distinct fashion by acetylation/ deacetylation, suggesting an integrated regulation mechanism. Overall, our study highlights the importance of protein acetylation for DNA replication initiation and progression, and suggests that SIRT1 may mediate a crosstalk between cellular circuits controlling metabolism and DNA synthesis.

SLFN11 is a recently discovered protein with a putative DNA/RNA helicase function. First identified in association with the maturation of thymocytes, SLFN11 was later causally associated, by two independent groups, with the resistance to DNA damaging agents such as topoisomerase I and II inhibitors, platinum compounds, and other alkylators, making it an attractive molecule for biomarker development. Later, SLFN11 was linked to antiviral response in human cells and interferon production, establishing a potential bond between immunity and chemotherapy. Recently, we demonstrated the potential role of SLN11 as a biomarker to predict sensitivity to the carboplatin/taxol combination in ovarian cancer. The present manuscript reports on the first international monothematic workshop on SLFN11. Several researchers from around the world, directly and actively involved in the discovery, functional characterization, and study of SLFN11 for its biomarker and medicinal properties gathered to share their views on the current knowledge advances concerning SLFN11. The aim of the manuscript is to summarize the authors’ interventions and the main take-home messages resulting from the workshop.