Concept Of Synthetic Lethality Research Articles

Inactivation of VRK1 sensitizes ovarian cancer to PARP inhibition through regulating DNA-PK stability

Ovarian cancer is the leading cause of gynecologic cancer death. Among the most innovative anti-cancer approaches, the genetic concept of synthetic lethality is that mutations in multiple genes work synergistically to effect cell death. Previous studies found that although vaccinia-related kinase-1 (VRK1) associates with DNA damage repair proteins, its underlying mechanisms remain unclear. Here, we found high VRK1 expression in ovarian tumors, and that VRK1 depletion can significantly promote apoptosis and cell cycle arrest. The effect of VRK1 knockdown on apoptosis was manifested by increased DNA damage, genomic instability, and apoptosis, and also blocked non-homologous end joining (NHEJ) by destabilizing DNA-PK. Further, we verified that VRK1 depletion enhanced sensitivity to a PARP inhibitor (PARPi), olaparib, promoting apoptosis through DNA damage, especially in ovarian cancer cell lines with high VRK1 expression. Proteins implicated in DNA damage responses are suitable targets for the development of new anti-cancer therapeutic strategies, and their combination could represent an alternative form of synthetic lethality. Therefore, normal protective DNA damage responses are impaired by combining olaparib with elimination of VRK1 and could be used to reduce drug dose and its associated toxicity. In summary, VRK1 represents both a potential biomarker for PARPi sensitivity, and a new DDR-associated therapeutic target, in ovarian cancer.

BRCA1/2 alterations and reversion mutations in the area of PARP inhibitors in high grade ovarian cancer: state of the art and forthcoming challenges.

BRCA1/2 genes are part of homologous recombination (HR) DNA repair pathways in charge of error-free double-strand break (DSB) repair. Loss-of-function mutations of BRCA1/2 genes have been associated for a long time with breast and ovarian cancer hereditary syndrome. Recently, polyadenosine diphosphate-ribose polymerase inhibitors (PARPi) have revolutionized the therapeutic landscape of BRCA1/2-mutated tumors, especially of BRCA1/2 high-grade serous ovarian cancer (HGSC), taking advantage of HR deficiency through the synthetic lethality concept. However, PARPi efficiency differs among patients, and most of them will develop resistance, particularly in the relapse setting. In the current proposal, we aim to review primary and secondary resistance to PARPi in HGSC owing to BRCA1/2 alterations. Of note, as several mechanisms of primary or secondary resistance to PARPi have been described, BRCA1/2 reversion mutations that restore HR pathways are by far the most reported. First, the type and location of the BRCA1/2 primary mutation have been associated with PARPi and platinum-salt sensitivity and impact the probability of the occurrence and the type of secondary reversion mutation. Furthermore, the presence of multiple reversion mutations and the variation of allelic frequency under treatment underline the role of intratumor heterogeneity (ITH) in treatment resistance. Of note, circulating tumor DNA might help us to detect and characterize reversion mutations and ITH to finally refine the treatment strategy. Importantly, forthcoming therapeutic strategies, including combination with antiangiogenics or with targeted therapies, may help us delay and overcome PARPi resistance secondary to BRCA1/2 reversion mutations. Also, progression despite PARPi therapy does not preclude PARPi rechallenge in selected patients.

Complex synthetic lethality in cancer.

The concept of synthetic lethality has been widely applied to identify therapeutic targets in cancer, with varying degrees of success. The standard approach normally involves identifying genetic interactions between two genes, a driver and a target. In reality, however, most cancer synthetic lethal effects are likely complex and also polygenic, being influenced by the environment in addition to involving contributions from multiple genes. By acknowledging and delineating this complexity, we describe in this article how the success rate in cancer drug discovery and development could be improved.

Functional inhibition of RECQL5 helicase elicits non-homologous end joining response and sensitivity of breast cancers to PARP inhibitor.

Poly (ADPRibose) Polymerase inhibitor (PARPi) are clinically approved for the treatment of BRCA-mutated hereditary breast and ovarian cancers with homologous recombination (HR) deficiency, based on synthetic lethality concept. However, ∼90% of breast cancers are BRCA-wild type; they repair PARPi mediated damage through HR, leading to intrinsic de novo resistance. Hence, there is an unmet need of exploring novel targets in HR-proficient aggressive breast cancers for PARPi treatment. RECQL5 physically interacts and disrupts RAD51 from pre-synaptic filaments, aiding HR resolution, replication fork protection and preventing illegitimate recombination. In the current investigation, we show that targeted inhibition of HR by stabilization of RAD51-RECQL5 complex by a pharmacological inhibitor of RECQL5 (4a; 1,3,4-oxadiazole derivative) in the presence of PARPi [talazoparib (BMN673)] leads to abolition of functional HR with uncontrolled activation of NHEJ repair. This was assessed by GFP based NHEJ reporter assay, KU80 recruitment and in vitro NHEJ based plasmid ligation assay. Concomitant treatment with talazoparib and 4a generates copious amounts of replication stress, prolonged cell cycle arrest, extensive double strand breaks (DSBs) and mitotic catastrophe, leading to sensitization of HR-proficient breast cancers. Suppression of NHEJ activity abolishes 4a-mediated sensitization of breast cancers to PARPi treatment. Imperatively, 4a was ineffective against normal mammary epithelial cells, which expresses low RECQL5 vis-à-vis breast cancer cells. Moreover, functional inhibition of RECQL5 suppresses metastatic potential of breast cancer cells in response to PARPi. Together, we identified RECQL5 as a novel pharmacological target for expanding PARPi based treatment horizon for HR-proficient cancers.

Homologous Recombination Deficiency (HRD) in Cutaneous Oncology

Skin cancers, including basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (SCC), and melanoma, are the most common malignancies in the United States. Loss of DNA repair pathways in the skin plays a significant role in tumorigenesis. In recent years, targeting DNA repair pathways, particularly homologous recombination deficiency (HRD), has emerged as a potential therapeutic approach in cutaneous malignancies. This review provides an overview of DNA damage and repair pathways, with a focus on HRD, and discusses major advances in targeting these pathways in skin cancers. Poly(ADP-ribose) polymerase (PARP) inhibitors have been developed to exploit HRD in cancer cells. PARP inhibitors disrupt DNA repair mechanisms by inhibiting PARP enzymatic activity, leading to the accumulation of DNA damage and cell death. The concept of synthetic lethality has been demonstrated in HR-deficient cells, such as those with BRCA1/2 mutations, which exhibit increased sensitivity to PARP inhibitors. HRD assessment methods, including genomic scars, RAD51 foci formation, functional assays, and BRCA1/2 mutation analysis, are discussed as tools for identifying patients who may benefit from PARP inhibitor therapy. Furthermore, HRD has been implicated in the response to immunotherapy, and the combination of PARP inhibitors with immunotherapy has shown promising results. The frequency of HRD in melanoma ranges from 18% to 57%, and studies investigating the use of PARP inhibitors as monotherapy in melanoma are limited. Further research is warranted to explore the potential of PARP inhibition in melanoma treatment.

Tumor sensitization to PARP inhibitors by DNA damaging synthetically lethal acylfulvenes.

e15123 Background: 20-30% of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor some level of deficiency in DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DNA damaging agents. The concept of synthetic lethality has been leveraged to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful in Homologous Recombination Deficiency (HRD) cancers. LP-100 and LP-184 are acylfulvene (AF) class small molecule prodrugs that are metabolized to active compounds by prostaglandin reductase 1 (PTGR1) that is often elevated in solid tumors. AF-induced DNA lesions are likely selectively processed by repair pathways including BRCA1/2 mediated Homologous Recombination (HR). Methods: Cell Titer Glo based 10 day cell viability assays were used to generate drug IC50 values in vitro. Bliss synergy scores were computed with scores ≥ 10 reflecting synergy. Drug-induced DNA double strand breaks (DSB) were detected and quantified with STRIDE (SensiTive Recognition of Individual DNA Ends) assay in cancer cells. A subcutaneous tumor xenograft model was used to determine tumor volumes in control and drug treated mice. Results: In a BRCA2 mutant ovarian cancer cell line OVCAR3, LP-184 and olaparib co-treatment in vitro showed synergistic effects (Bliss score 10). Similarly in a BRCA2 mutant prostate cancer cell line 22Rv1, LP-100 combination with rucaparib was synergistic (Bliss score 18). 400 nM LP-184 showed a time dependent increase in DNA DSB signals in vitro, 1.7X more so in BRCA2 mutant vs wild type (WT) DLD-1 isogenic colon cancer cell lines up to 24 hours. To validate the hypothesis that AF-induced DSBs remain unresolved if exposed to and further synergize with PARPi in vivo, we tested LP-100 plus olaparib in a DU145 (BRCA2 mutant) prostate cancer xenograft model. LP-100 + olaparib treatment resulted in sustained, durable tumor regression indicating in vivo synergy whereas either agent alone did not produce tumor shrinkage. Day 42 tumor growth inhibition was 66% for 2.5 mg/kg LP-100 alone, 34% for 40 mg/kg olaparib alone and 108% for the combination. Moreover, LP-184 treatment resulted in early onset and complete tumor regression in 7 PDX models of PARPi resistant triple negative breast cancers (TNBC) harboring BRCA1/2 loss whereas olaparib/ niraparib remained entirely ineffective in these. Conclusions: AF response is influenced by tumor DNA repair competence. LP-100 and LP-184 are synthetically lethal resulting from their abilities to cause unresolvable DNA damage where tumor cells express high PTGR1 and are deficient in the HR pathway. LP-184 single agent was superior to PARPi in TNBC and LP-100 combination made an olaparib resistant tumor highly sensitive, thereby facilitating a path to extend the opportunities for both AFs and PARPis and enhance the clinical utilization of PARPi.

Investigation of synthetic lethality gene pairs in the DNA damage response pathway in response to immunotherapy in pan-cancer.

2567 Background: The concept of synthetic lethality (SL) refers to the phenomenon where the simultaneous loss of function of two genes leads to cell death, while the loss of function of either gene alone has no effect. PARP inhibitors target poly ADP ribose polymerase (PARP) enzymes, which are involved in DNA repair, are the successful cases of utilizing synthetic lethality-based therapy to treat cancers with DNA damage response (DDR) deficiency. The prognosis for cancer patients already has a combination of synthetic lethal mutations especially in DDR pathway can vary and it is not yet fully understood how these mutations may impact the response to immunotherapy. Methods: SL gene combinations in the DDR pathway were screened out from SynLethDB database (http://histone.sce.ntu.edu.sg/SynLethDB/). The immunotherapy cohort (Samstein 2019, n=1661) and non-immunotherapy cohort (Zehir 2017, n=10336) with solid tumor were used to Kaplan-Meier analyze the correlation between SL gene combinations and prognosis. TMB scores were compared between DDR co-mutation group and non DDR co-mutation group. For investigating the associations between immune infiltrates and genetic features, TIMER 2.0 database ( http://timer.cistrome.org/ ) was conducted to estimation of immune infiltration levels on DDR mutation genes in pan-cancer. Results: 430 pairs of DDR mutation combinations were used to subgroup stratification, the DDR co-mutation group was associated with worse prognosis in non-immunotherapy cohort (HR=1.305, p<0.0001) but better in immunotherapy cohort (HR=0.741,p<0.0061). Interestingly, the TMB score in the DDR co-mutation group was significantly higher than non DDR co-mutation group (mean 50.03 vs 8.868, p<0.0001) in immunotherapy cohort. The high-frequency mutation combinations (>10%) were ATM-POLE(14.40%), ATM-ATR (13.60%), ATR-BRCA2 (13.60%), ATM-BRCA2 (12.00%) and BRCA2-RECQL4 (10.40%), respectively. Comparing common immune component with statistically significant under different DDR mutant genes, the ATM-POLE co-mutation tumor tended to have more CD4+ T cell infiltration while the ATM-ATR co-mutation tumor appeared to have more myeloid dendritic cell infiltration. Conclusions: The combinations of DDR SL mutation tumor might result to worse prognosis whereas these patients might benefit from immunotherapy. Our results will help to shed light on the complex interplay for the combinations of SL mutation in DDR pathway between cancer cells and the immune system and provide new insights into the development of effective cancer treatments.

Clinical efficacy of PARP inhibitors in breast cancer

BRCA1 and BRCA2 are key tumor suppressor genes that are essential for the homologous recombination DNA repair pathway. Loss of function mutations in these genes result in hereditary breast and ovarian cancer syndromes, which comprise approximately 5% of cases. BRCA1/2 mutations are associated with younger age of diagnosis and increased risk of recurrences. The concept of synthetic lethality led to the development of PARP inhibitors which cause cell cytotoxicity via the inhibition of PARP1, a key DNA repair protein, in cells with germline BRCA1/2 mutations. Although still poorly understood, the most well-acknowledged proposed mechanisms of action of PARP1 inhibition include the inhibition of single strand break repair, PARP trapping, and the upregulation of non-homologous end joining. Olaparib and talazoparib are PARP inhibitors that have been approved for the management of HER2-negative breast cancer in patients with germline BRCA1/2 mutations. This review article highlights the clinical efficacy of PARP inhibitors in patients with HER2-negative breast cancer in early and advanced settings.

Abstract 6241: LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in DNA damage repair deficient cancers

Abstract As many as one third of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor deficiency in some DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DDR inhibitors. The concept of synthetic lethality has been successfully applied to develop strategies to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful particularly in Homologous Recombination Deficiency (HRD) cancers. Other agents have also been designed to target these DDR deficiencies in cancer cells and have potential to be clinically beneficial. LP-184 is a next generation acylfulvene prodrug metabolized to an active compound by prostaglandin reductase 1 (PTGR1) that is often elevated in multiple tumors. LP-184-induced DNA lesions are likely repaired by the Nucleotide Excision Repair (NER) and Homologous Repair (HR) pathways. Reduced activities of multiple DDR-related pathways in solid tumor models correlated with increased sensitivities to LP-184 as indicated by Gene Set Enrichment Analysis. Chinese Hamster Ovary cells with NER mutations (ERCC1/2/6) were at least 4 fold more sensitive to LP-184 relative to otherwise isogenic parental cells. ERCC2 or BRCA2 depletion enhanced LP-184 sensitivity 5-8X in a metastatic prostate cancer cell line. LP-184 showed ~120 fold higher potency (IC50 77 nM) compared to Olaparib in HRD prostate cancer organoid model LuCaP 96 (BRCA2/CHEK2 inactivating mutations). Across 14 patient-derived pancreatic, lung and prostate tumor models harboring HR mutations (BRCA1/2, CHEK1/2, ATM/ATR, PALB2, PARP1/2, RAD51, FANCA/B), LP-184 showed ex vivo IC50s in the range of 30-300 nM, whereas Olaparib IC50s ranged from 1700-6900 nM. LP-184 treatment resulted in complete tumor regression (107-141% TGI) in 10/10 HR deficient triple negative breast cancer PDX models of which 7/10 were resistant Olaparib/Niraparib and to Doxorubicin/Cyclophosphamide. LP-184 treatment resulted in complete and durable tumor regression in HRD (BRCA1/ATR mutant) pancreatic cancer PDX models (112-140% TGI). In the ovarian cancer cell line OVCAR3 having HRD/NERD phenotype, LP-184 IC50 was 13 nM, Olaparib IC50 was 145 nM and their combination Bliss Synergy Score was 16 indicating synergy. LP-184 is synthetic lethal resulting from its abilities to cause unresolvable DNA damage if tumor cells express high PTGR1 and are deficient in NER/HR genes. Unlike PARPi, LP-184 has striking activity in both NERD as well as HRD cancers. These strong data have prompted the development of a soon to be launched clinical trial to translate the broad preclinical anticancer activity of LP-184 in solid tumors with HR/NER pathway defects, such as pancreatic, prostate, ovarian and breast cancers. Citation Format: Aditya Kulkarni, Jianli Zhou, Neha Biyani, Kishor Bhatia, Panna Sharma. LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in DNA damage repair deficient cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6241.

Abstract 6187: The combination of Trifluridine/Tipiracil and a WEE1 inhibitor is an effective and tolerable candidate strategy against ESCC

Abstract Background: Recently we reported that trifluridine (FTD)/Tipiracil (TPI) is tolerable for unresectable Esophageal squamous cell carcinoma (ESCC) patients although the anti-tumor effect was modest (Mori Y, et al. Esophagus 2022). Therefore, we aimed at developing a combination therapy with FTD/TPI with another small molecule to achieve better efficacy against ESCC. CHK1 inhibitor was considered to be the candidate, because combination treatment with FTD/TPI and prexasertib showed potent antitumor effects in p53-mutant ESCC cells through the synthetic lethality (Ohashi S, et al. Mol Cancer Ther 2020); however, CHK1 inhibitors are not clinically available for further clinical development. Here we explored the concept of synthetic lethality with CHK1 perturbation to the whole ATR-CHK1-WEE1 pathway, especially to the downward WEE1 which directly targets a cell cycle regulator CDK1. The aim of this study is to elucidate the efficacy of the combination of a WEE1 inhibitor (WEE1i) MK1775 with FTD/TPI in ESCC. Methods: ESCC cells (TE-8 and TE-11) are used for in vitro assay, with compounds including FTD, MK1775 (WEE1i). Mitosis assay (flowcytometry with phospho-Histone H3 [p-hH3] antibody), cell viability assay (WST-1 and clonogenic assays), cytotoxicity assay (CytoTox-Glo assay), and western blotting (double-strand DNA break [γ-H2AX], DDR activity [phospho-CHK1], and CDK1 activity [phospho-Tyr15-CDK1]) were performed. Antitumor effects and tolerability were observed in vivo with TE-8 xenograft model with nu/nu nude mice. Results: FTD induced activation of CHK1 and inhibition of CDK1 sequentially in ESCC cells. FTD also decreased the proportion of p-hH3 positive cells in mitosis assay. Combination treatment with WEE1i and FTD had activated CDK1, increased p-hH3 positive cells, and induced γ-H2AX. The WST-1 cell viability assay showed significant sensitizing effect of WEE1i to FTD in ESCC cells. The cytotoxicity assay also revealed the significant increase of dead cells by the combination treatment (p = 0.003). Furthermore, we confirmed the combination treatment significantly suppressed xenografted tumor growth (-86%) without major adverse events in vivo (two-way ANOVA and Tukey post-hoc analyses: FTD/TPI vs. control, P < 0.05; MK1775 vs. control, P < 0.05, without significant interaction between the FTD/TPI treatment and MK1775 treatment). Conclusion: FTD/TPI and WEE1i combination showed potent cytotoxicity, and is considered as a candidate treatment strategy against ESCC. Citation Format: Hoang Trang Nguyen Vu, Osamu Kikuchi, Tomoki Saito, Yukie Nakai, Tomomi Ida, Yuki Kondo, Shigeki Kataoka, Yosuke Mitani, Shinya Ohashi, Manabu Muto. The combination of Trifluridine/Tipiracil and a WEE1 inhibitor is an effective and tolerable candidate strategy against ESCC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6187.

Abstract 6108: Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells

Abstract Since the early days of the synthetic lethality concept in DNA Damage Response the status of homologous recombination (HR) repair in cancer cells have been the focus of attention of researchers and clinicians. While different approaches exist, such as the RAD51 immunofluorescence (IF) or HRD genomic assays, functional biomarkers that can assess HR proficiency are missing. We report here the development, optimization and validation of two complementary, HR-specific functional assays. The assays, which are based on the STRIDE platform technology, detect double-strand DNA breaks localized in close proximity to RPA or RAD51 proteins. The optimization phase of assay development was performed in U2OS cells. First, repeatability (intra-run variation) and reproducibility (inter-run variation) of the assays were measured in untreated cells. Then, a series of technical negative controls was performed which have shown that the number of false-positive readouts is below 10% of the total number of signals. Finally, treatment of cells with compounds known to induce double-strand DNA breaks (etoposide and cisplatin) resulted in statistically significant increase in the number of detected dSTRIDE-RAD51 and dSTRIDE-RPA foci when compared to untreated controls. The assays were further validated in NCI-H661 (BRCA2 wild-type) and NCI-H169 (BRCA2 KO) cell line pair. The cells were treated with two concentrations of etoposide and the readouts from dSTRIDE, detecting the total pool of DSBs and dSTRIDE-HR assays were compared. In NCI-H661 cells, treatment with etoposide resulted in an increase in the number of double-strand breaks detected by dSTRIDE and as expected, more DSBs were formed after treatment with the higher concentration. dSTRIDE-HR assays confirmed that approximately 15% and 10% of these DSBs contain RPA and RAD51 proteins, respectively. In NCI-H169 cells etoposide produced a stronger reaction with even more DSBs detected by dSTRIDE, but importantly, no increase in the number of dSTRIDE-RAD51 foci was observed. dSTRIDE-RPA foci increased after treatment hinting that this step of HR remains unperturbed. Interestingly, the number of dSTRIDE-RAD51 foci in untreated cells was comparable between the two cell lines. In conclusion, we show here that two newly developed dSTRIDE-HR assays are well validated and can be successfully applied to report on the status of homologous recombination repair in different cell models. Citation Format: Kamil Solarczyk, Agnieszka Waligórska, Karolina Uznańska, Zsombor Prucsi, Olga Wójcikowska, Ewelina Matuszyk, Magdalena Bartyńska, Agata Kitlińska, Aleksandra Bober, Franek Sierpowski, Maja Białecka, Monika Jarosz, Malgorzata Szczygiel, Szymon Koman, Karolina Korpanty, Lukasz Beben, Lukasz Bandzarewicz, Przemyslaw Stachura, Magdalena Kordon-Kiszala. Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6108.

PARP inhibitors - A better selection of patients

PARP inhibitors (PARPi) have established themselves as a class of essential anti-cancer drugs. They inhibit PARP proteins involved in DNA damage repair. Their anti-tumor action requires a concomitant abnormality in DNA damage repair, the homologous recombination deficiency (HRD). The genomic instability being too substantial, the tumor cell goes into apoptosis (concept of synthetic lethality). This last decade, the selection of patients benefiting from PARPi has been refined with convincing results for ovarian cancers, but also breast, prostate and pancreatic cancers. This article presents recent data that have impacted our clinical practice and the PARPi authorized in Switzerland.

Discovery of synthetic lethal interactions from large-scale pan-cancer perturbation screens

The development of cancer therapies is limited by the availability of suitable drug targets. Potential candidate drug targets can be identified based on the concept of synthetic lethality (SL), which refers to pairs of genes for which an aberration in either gene alone is non-lethal, but co-occurrence of the aberrations is lethal to the cell. Here, we present SLIdR (Synthetic Lethal Identification in R), a statistical framework for identifying SL pairs from large-scale perturbation screens. SLIdR successfully predicts SL pairs even with small sample sizes while minimizing the number of false positive targets. We apply SLIdR to Project DRIVE data and find both established and potential pan-cancer and cancer type-specific SL pairs consistent with findings from literature and drug response screening data. We experimentally validate two predicted SL interactions (ARID1A-TEAD1 and AXIN1-URI1) in hepatocellular carcinoma, thus corroborating the ability of SLIdR to identify potential drug targets.

Systematic analysis of cancer-specific synthetic lethal interactions provides insight into personalized anticancer therapy.

The concept of synthetic lethality has great potential for anticancer therapy as a new strategy to specifically kill cancer cells while sparing normal cells. To further understand the potential molecular interactions and gene characteristics involved in synthetic lethality, we performed a comprehensive analysis of predicted cancer-specific genetic interactions. Many genes were identified as cancer-associated genes that contributed to multiple biological processes and pathways, and the gene features were not random, indicating their potential roles in human carcinogenesis. Some relevant genes detected in multiple cancers were prone to be enriched in specific biological progresses and pathways, especially processes associated with DNA damage, chromosome-related functions and cancer pathways. These findings strongly implicated potential roles for these genes in cancer pathophysiology and functional relationships, as well as applications for future anticancer drug discovery. Further experimental validation indicated that the synthetic lethal interaction of APC and GFER may provide a potential anticancer strategy for patients with APC-mutant colon cancer. These results will contribute to further exploration of synthetic lethal interactions and broader application of the concept of synthetic lethality in anticancer therapeutics.

A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.

BRCA Mutations in Ovarian and Prostate Cancer: Bench to Bedside.

Simple SummaryDNA damage is one of the hallmarks of cancer. Epithelial ovarian cancer (EOC) —especially the high-grade serous subtype—harbors a defect in at least one DNA damage response (DDR) pathway. Defective DDR results from a variety of lesions affecting homologous recombination (HR) and nonhomologous end joining (NHEJ) for double strand breaks, base excision repair (BER), and nucleotide excision repair (NER) for single strand breaks and mismatch repair (MMR). Apart from the EOC, mutations in the DDR genes, such as BRCA1 and BRCA2, are common in prostate cancer as well. Among them, BRCA2 lesions are found in 12% of metastatic castration-resistant prostate cancers, but very rarely in primary prostate cancer. Better understanding of the DDR pathways is essential in order to optimize the therapeutic choices, and has led to the design of biomarker-driven clinical trials. Poly(ADP-ribose) polymerase (PARP) inhibitors are now a standard therapy for EOC patients, and more recently have been approved for the metastatic castration-resistant prostate cancer with alterations in DDR genes. They are particularly effective in tumours with HR deficiency.DNA damage repair (DDR) defects are common in different cancer types, and these alterations can be exploited therapeutically. Epithelial ovarian cancer (EOC) is among the tumours with the highest percentage of hereditary cases. BRCA1 and BRCA2 predisposing pathogenic variants (PVs) were the first to be associated with EOC, whereas additional genes comprising the homologous recombination (HR) pathway have been discovered with DNA sequencing technologies. The incidence of DDR alterations among patients with metastatic prostate cancer is much higher compared to those with localized disease. Genetic testing is playing an increasingly important role in the treatment of patients with ovarian and prostate cancer. The development of poly (ADP-ribose) polymerase (PARP) inhibitors offers a therapeutic strategy for patients with EOC. One of the mechanisms of PARP inhibitors exploits the concept of synthetic lethality. Tumours with BRCA1 or BRCA2 mutations are highly sensitive to PARP inhibitors. Moreover, the synthetic lethal interaction may be exploited beyond germline BRCA mutations in the context of HR deficiency, and this is an area of ongoing research. PARP inhibitors are in advanced stages of development as a treatment for metastatic castration-resistant prostate cancer. However, there is a major concern regarding the need to identify reliable biomarkers predictive of treatment response. In this review, we explore the mechanisms of DDR, the potential for genomic analysis of ovarian and prostate cancer, and therapeutics of PARP inhibitors, along with predictive biomarkers.

Identifying Lethal Dependencies with HUGE Predictive Power.

Simple SummaryThis work shows that the predictions of lethal dependencies (LEDs) between genes can be dramatically improved by incorporating the “HUb effect in Genetic Essentiality” (HUGE) of gene alterations. In three genome-wide loss-of-function screens—Project Score, CERES score and DEMETER score—LEDs are identified with 75 times larger statistical power than using state-of-the-art methods. In AML, we identified LEDs not recalled by previous pipelines, including FLT3-mutant genotypes sensitive to FLT3 inhibitors. Interestingly, in-vitro validations confirm lethal de-pendencies of either NRAS or PTPN11 depending on the NRAS mutational status.Recent functional genomic screens—such as CRISPR-Cas9 or RNAi screening—have fostered a new wave of targeted treatments based on the concept of synthetic lethality. These approaches identified LEthal Dependencies (LEDs) by estimating the effect of genetic events on cell viability. The multiple-hypothesis problem is related to a large number of gene knockouts limiting the statistical power of these studies. Here, we show that predictions of LEDs from functional screens can be dramatically improved by incorporating the “HUb effect in Genetic Essentiality” (HUGE) of gene alterations. We analyze three recent genome-wide loss-of-function screens—Project Score, CERES score and DEMETER score—identifying LEDs with 75 times larger statistical power than using state-of-the-art methods. Using acute myeloid leukemia, breast cancer, lung adenocarcinoma and colon adenocarcinoma as disease models, we validate that our predictions are enriched in a recent harmonized knowledge base of clinical interpretations of somatic genomic variants in cancer (AUROC > 0.87). Our approach is effective even in tumors with large genetic heterogeneity such as acute myeloid leukemia, where we identified LEDs not recalled by previous pipelines, including FLT3-mutant genotypes sensitive to FLT3 inhibitors. Interestingly, in-vitro validations confirm lethal dependencies of either NRAS or PTPN11 depending on the NRAS mutational status. HUGE will hopefully help discover novel genetic dependencies amenable for precision-targeted therapies in cancer. All the graphs showing lethal dependencies for the 19 tumor types analyzed can be visualized in an interactive tool.

Targeting euchromatic histone lysine methyltransferases sensitizes colorectal cancer to histone deacetylase inhibitors.

Epigenetic dysregulation is an important feature of colorectal cancer (CRC). Combining epigenetic drugs with other antineoplastic agents is a promising treatment strategy for advanced cancers. Here, we exploited the concept of synthetic lethality to identify epigenetic targets that act synergistically with histone deacetylase (HDAC) inhibitors to reduce the growth of CRC. We applied a pooled CRISPR-Cas9 screen using a custom sgRNA library directed against 614 epigenetic regulators and discovered that knockout of the euchromatic histone-lysine N-methyltransferases 1 and 2 (EHMT1/2) strongly enhanced the antiproliferative effect of clinically used HDAC inhibitors. Using tissue microarrays from 1066 CRC samples with different tumor stages, we showed that low EHMT2 protein expression is predominantly found in advanced CRC and associated with poor clinical outcome. Cotargeting of HDAC and EHMT1/2 with specific small molecule inhibitors synergistically reduced proliferation of CRC cell lines. Mechanistically, we used a high-throughput Western blot assay to demonstrate that both inhibitors elicited distinct cellular mechanisms to reduce tumor growth, including cell cycle arrest and modulation of autophagy. On the epigenetic level, the compounds increased H3K9 acetylation and reduced H3K9 dimethylation. Finally, we used a panel of patient-derived CRC organoids to show that HDAC and EHMT1/2 inhibition synergistically reduced tumor viability in advanced models of CRC.

PARP Inhibitors: A New Horizon for Patients with Prostate Cancer.

The introduction of PARP inhibitors (PARPi) in prostate cancer is a milestone and provides a pathway to hope in fighting this disease. It is the first time that drugs, based on the concept of synthetic lethality, have been approved for prostate cancer. In addition, it is also the first time that genetic mutation tests have been included in the therapeutic algorithm of this disease, representing a significant step forward for precision and personalized treatment of prostate cancer. The objectives of this review are: (1) understanding the mechanism of action of PARPi in monotherapy and combinations; (2) gaining insights on patient selection for PARPi; (3) exposing the pivotal studies that have allowed its approval, and; (4) offering an overview of the ongoing trials. Nevertheless, many unsolved questions remain, such as the number of patients who could potentially benefit from PARPi, whether to use PARPi in monotherapy or in combination, and when is the best time to use them in advanced or localized disease. To answer these and other questions, many clinical trials are underway. Some of them have recently demonstrated promising results that may favor the introduction of new combinations in metastatic castration-resistant prostate cancer.

Abstract 1918: A novel computational framework predicts synthetic lethal interactions between key regulators of the DNA damage response and chromatin modifiers

Abstract The concept of synthetic lethality (SL) has made a pivotal impact for the development of the anti-cancer drug olaparib, the first approved agent targeting the DNA Damage Response (DDR). Typically, SL is described as the interaction of two genes, whereby simultaneous inactivation of both genes results in cell death whereas loss of one gene can be tolerated. Given the importance of SL to develop highly selective anti-cancer therapeutics, large efforts have been undertaken to identify these interactions, both experimentally and computationally. Here, we present a novel computational framework harnessing large-scale cell line gene inactivation screens (DepMap, Project Score), as well as patient data (TCGA), to discover known and novel SL gene pairs. Overall, we implemented six statistical tests considering gene dependency scores, genomic profiles, gene expression and patient survival as parameters. We further utilized data from public drug screening consortia to validate our top-ranking pairs. We applied our framework to a defined target space covering genes relating to DDR, chromatin binding, cell cycle and druggable genes (overall > 2.5 M. pairs were tested). When focusing on SL partners for three DDR genes with promising inhibitors in early clinical development: ATM, ATR and DNA-PK; we noticed that chromatin modifiers were enriched in the top ranking pairs. In particular, SL interactions were predicted with histone (de)methylases, histone (de)acetyltransferases and members of SWI/SNF family. For instance, we observed that loss of function mutations in several members of the KMT2 family, also known as MLL family, would render cancers cells more dependent on ATR or ATM. Similarly, drug sensitivity was increased for selected DDR inhibitors in cell lines with KMT2 mutations. Members of the KMT2 family play essential roles in transcription, but have recently also been shown to be recruited to DNA damage sites and may be mediators for PARP inhibitor sensitivity. The KMT2 family is frequently mutated in several cancers types such as endometrial and bladder, thus underpinning their suitability as potential selection biomarkers as well as relevance to cancer. Furthermore, we observed that patients with mutations in EP300, a histone lysine acetyl transferase, exhibited an increase in ATR expression which may comprise a compensatory mechanism. In addition, patients with ATR and EP300 double mutation tend to have a better probability of survival, suggesting decreased tumor fitness. In summary, we present a modular framework to predict SL gene pairs to a defined target space. Here, we focused on discovering novel SL relationships for the key DDR regulators ATM, ATR and DNA-PK. Our results provide not only new biomarker hypotheses for further validation, but also suggest that cancers with a high mutation rate in chromatin modifying genes may be efficiently targeted by DDRi. Citation Format: Anna M. Coenen-Stass, Magda Markowska, Magdalena Budzinska-Zaniewska, Krzysztof Kolmus, Ewa Szczurek, Eike Staub. A novel computational framework predicts synthetic lethal interactions between key regulators of the DNA damage response and chromatin modifiers [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 1918.