Randomized Phase II Study of Gemcitabine With or Without ATR Inhibitor Berzosertib in Platinum-Resistant Ovarian Cancer: Final Overall Survival and Biomarker Analyses.

PURPOSE: The multicenter, open-label, randomized phase 2 NCI-9944 study (NCT02595892) demonstrated that addition of ATR inhibitor (ATRi) berzosertib to gemcitabine increased progression-free survival (PFS) compared to gemcitabine alone (hazard ratio [HR]=0.57, one-sided log-rank P = .044, which met the one-sided significance level of 0.1 used for sample size calculation). METHODS: We report here the final overall survival (OS) analysis and biomarker correlations (ATM expression by immunohistochemistry, mutational signature 3 and a genomic biomarker of replication stress) along with post-hoc exploratory analyses to adjust for crossover from gemcitabine to gemcitabine/berzosertib. RESULTS: At the data cutoff of January 27, 2023 (>30 months of additional follow-up from the primary analysis), median OS was 59.4 weeks with gemcitabine/berzosertib versus 43.0 weeks with gemcitabine alone (HR 0.79, 90% CI 0.52 to 1.2, one-sided log-rank P = .18). An OS benefit with addition of berzosertib to gemcitabine was suggested in patients stratified into the platinum-free interval ≤3 months (N = 26) subgroup (HR, 0.48, 90% CI 0.22 to 1.01, one-sided log-rank P =.04) and in patients with ATM-negative/low (N = 24) tumors (HR, 0.50, 90% CI 0.23 to 1.08, one-sided log-rank P = .06). CONCLUSION: The results of this follow-up analysis continue to support the promise of combined gemcitabine/ATRi therapy in platinum resistant ovarian cancer, an active area of investigation with several ongoing clinical trials.

Biological basis of extensive pleiotropy between blood traits and cancer risk.

BACKGROUND: The immune system has a central role in preventing carcinogenesis. Alteration of systemic immune cell levels may increase cancer risk. However, the extent to which common genetic variation influences blood traits and cancer risk remains largely undetermined. Here, we identify pleiotropic variants and predict their underlying molecular and cellular alterations. METHODS: Multivariate Cox regression was used to evaluate associations between blood traits and cancer diagnosis in cases in the UK Biobank. Shared genetic variants were identified from the summary statistics of the genome-wide association studies of 27 blood traits and 27 cancer types and subtypes, applying the conditional/conjunctional false-discovery rate approach. Analysis of genomic positions, expression quantitative trait loci, enhancers, regulatory marks, functionally defined gene sets, and bulk- and single-cell expression profiles predicted the biological impact of pleiotropic variants. Plasma small RNAs were sequenced to assess association with cancer diagnosis. RESULTS: The study identified 4093 common genetic variants, involving 1248 gene loci, that contributed to blood-cancer pleiotropism. Genomic hotspots of pleiotropism include chromosomal regions 5p15-TERT and 6p21-HLA. Genes whose products are involved in regulating telomere length are found to be enriched in pleiotropic variants. Pleiotropic gene candidates are frequently linked to transcriptional programs that regulate hematopoiesis and define progenitor cell states of immune system development. Perturbation of the myeloid lineage is indicated by pleiotropic associations with defined master regulators and cell alterations. Eosinophil count is inversely associated with cancer risk. A high frequency of pleiotropic associations is also centered on the regulation of small noncoding Y-RNAs. Predicted pleiotropic Y-RNAs show specific regulatory marks and are overabundant in the normal tissue and blood of cancer patients. Analysis of plasma small RNAs in women who developed breast cancer indicates there is an overabundance of Y-RNA preceding neoplasm diagnosis. CONCLUSIONS: This study reveals extensive pleiotropism between blood traits and cancer risk. Pleiotropism is linked to factors and processes involved in hematopoietic development and immune system function, including components of the major histocompatibility complexes, and regulators of telomere length and myeloid lineage. Deregulation of Y-RNAs is also associated with pleiotropism. Overexpression of these elements might indicate increased cancer risk.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1-autoinhibited for chromatin binding-that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

Targeting MCL-1 triggers DNA damage and an anti-proliferative response independent from apoptosis induction.

MCL-1 is a high-priority target due to its dominant role in the pathogenesis and chemoresistance of cancer, yet clinical trials of MCL-1 inhibitors are revealing toxic side effects. MCL-1 biology is complex, extending beyond apoptotic regulation and confounded by its multiple isoforms, its domains of unresolved structure and function, and challenges in distinguishing noncanonical activities from the apoptotic response. We find that, in the presence or absence of an intact mitochondrial apoptotic pathway, genetic deletion or pharmacologic targeting of MCL-1 induces DNA damage and retards cell proliferation. Indeed, the cancer cell susceptibility profile of MCL-1 inhibitors better matches that of anti-proliferative than pro-apoptotic drugs, expanding their potential therapeutic applications, including synergistic combinations, but heightening therapeutic window concerns. Proteomic profiling provides a resource for mechanistic dissection and reveals the minichromosome maintenance DNA helicase as an interacting nuclear protein complex that links MCL-1 to the regulation of DNA integrity and cell-cycle progression.

Dynamics of the DYNLL1-MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs.

The extent and efficacy of DNA end resection at DNA double-strand breaks (DSB) determine the repair pathway choice. Here we describe how the 53BP1-associated protein DYNLL1 works in tandem with the Shieldin complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1, where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1, predominantly in G1 cells. Shieldin localization to DSBs depends on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be resensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.

Serum miRNA-based signature indicates radiation exposure and dose in humans: A multicenter diagnostic biomarker study.

PURPOSE: Mouse and non-human primate models showed that serum miRNAs may be used to predict the biological impact of radiation doses. We hypothesized that these results can be translated to humans treated with total body irradiation (TBI), and that miRNAs may be used as clinically feasible biodosimeters. METHODS: To test this hypothesis, serial serum samples were obtained from 25 patients (pediatric and adults) who underwent allogeneic stem-cell transplantation and profiled for miRNA expression using next-generation sequencing. miRNAs with diagnostic potential were quantified with qPCR and used to build logistic regression models with lasso penalty to reduce overfitting, identifying samples drawn from patients who underwent total body irradiation to a potentially lethal dose. RESULTS: Differential expression results were consistent with previous studies in mice and non-human primates. miRNAs with detectable expression in this and two prior animal sets allowed for distinction of the irradiated from non-irradiated samples in mice, macaques and humans, validating the miRNAs as radiation-responsive through evolutionarily conserved transcriptional regulation mechanisms. Finally, we created a model based on the expression of miR-150-5p, miR-30b-5p and miR-320c normalized to two references and adjusted for patient age with an AUC of 0.9 (95%CI:0.83-0.97) for identifying samples drawn after irradiation; a separate model differentiating between high and low radiation dose achieved AUC of 0.85 (95%CI: 0.74-0.96). CONCLUSIONS: We conclude that serum miRNAs reflect radiation exposure and dose for humans undergoing TBI and may be used as functional biodosimeters for precise identification of people exposed to clinically significant radiation doses.

Identification of BRCA1/2 mutation female carriers using circulating microRNA profiles.

Identifying germline BRCA1/2 mutation carriers is vital for reducing their risk of breast and ovarian cancer. To derive a serum miRNA-based diagnostic test we used samples from 653 healthy women from six international cohorts, including 350 (53.6%) with BRCA1/2 mutations and 303 (46.4%) BRCA1/2 wild-type. All individuals were cancer-free before and at least 12 months after sampling. RNA-sequencing followed by differential expression analysis identified 19 miRNAs significantly associated with BRCA mutations, 10 of which were ultimately used for classification: hsa-miR-20b-5p, hsa-miR-19b-3p, hsa-let-7b-5p, hsa-miR-320b, hsa-miR-139-3p, hsa-miR-30d-5p, hsa-miR-17-5p, hsa-miR-182-5p, hsa-miR-421, hsa-miR-375-3p. The final logistic regression model achieved area under the receiver operating characteristic curve 0.89 (95% CI: 0.87-0.93), 93.88% sensitivity and 80.72% specificity in an independent validation cohort. Mutated gene, menopausal status or having preemptive oophorectomy did not affect classification performance. Circulating microRNAs may be used to identify BRCA1/2 mutations in patients of high risk of cancer, offering an opportunity to reduce screening costs.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1 - autoinhibited for chromatin binding - that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

Dynamics of the DYNLL1/MRE11 complex regulates DNA end resection and recruitment of the Shieldin complex to DSBs.

Extent and efficacy of DNA end resection at DNA double strand break (DSB)s determines the choice of repair pathway. Here we describe how the 53BP1 associated protein DYNLL1 works in tandem with Shieldin and the CST complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1 where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1 predominantly in the G1 cells. Shieldin localization to DSBs is dependent on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be re-sensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.

Targeting replication stress in cancer therapy.

Replication stress is a major cause of genomic instability and a crucial vulnerability of cancer cells. This vulnerability can be therapeutically targeted by inhibiting kinases that coordinate the DNA damage response with cell cycle control, including ATR, CHK1, WEE1 and MYT1 checkpoint kinases. In addition, inhibiting the DNA damage response releases DNA fragments into the cytoplasm, eliciting an innate immune response. Therefore, several ATR, CHK1, WEE1 and MYT1 inhibitors are undergoing clinical evaluation as monotherapies or in combination with chemotherapy, poly[ADP-ribose]polymerase (PARP) inhibitors, or immune checkpoint inhibitors to capitalize on high replication stress, overcome therapeutic resistance and promote effective antitumour immunity. Here, we review current and emerging approaches for targeting replication stress in cancer, from preclinical and biomarker development to clinical trial evaluation.

CCAR2 functions downstream of the Shieldin complex to promote double-strand break end-joining.

The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The Shieldin complex is a downstream effector of the 53BP1-RIF1 pathway. Here, we identify a component of this pathway, CCAR2/DBC1, which is also required for restriction of DNA end-resection. CCAR2 co-immunoprecipitates with the Shieldin complex, and knockout of CCAR2 in a BRCA1-deficient cell line results in elevated DSB end-resection, RAD51 loading, and PARP inhibitor (PARPi) resistance. Knockout of CCAR2 is epistatic with knockout of other Shieldin proteins. The S1-like RNA-binding domain of CCAR2 is required for its interaction with the Shieldin complex and for suppression of DSB end-resection. CCAR2 functions downstream of the Shieldin complex, and CCAR2 knockout cells have delayed resolution of Shieldin complex foci. Forkhead-associated (FHA)-dependent targeting of CCAR2 to DSB sites re-sensitized BRCA1-/-SHLD2-/- cells to PARPi. Taken together, CCAR2 is a functional component of the 53BP1-RIF1 pathway, promotes the refill of resected DSBs, and suppresses homologous recombination.

A druggable addiction to de novo pyrimidine biosynthesis in diffuse midline glioma.

Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer driven by oncohistones that do not readily lend themselves to drug development. To identify druggable targets for DMG, we conducted a genome-wide CRISPR screen that reveals a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. This metabolic vulnerability reflects an elevated rate of uridine/uracil degradation that depletes DMG cells of substrates for the alternate salvage pyrimidine biosynthesis pathway. A clinical stage inhibitor of DHODH (rate-limiting enzyme in the de novo pathway) diminishes uridine-5'-phosphate (UMP) pools, generates DNA damage, and induces apoptosis through suppression of replication forks-an "on-target" effect, as shown by uridine rescue. Matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy imaging demonstrates that this DHODH inhibitor (BAY2402234) accumulates in the brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in vivo, and prolongs survival of mice bearing intracranial DMG xenografts, highlighting BAY2402234 as a promising therapy against DMGs.

TOPBPing up DSBs with PARylation.

Zhao et al. (2022) demonstrate that HIV Tat-specific factor 1, an RPA PARylation reader, recruits Topoisomerase IIß-binding protein 1 to double-strand break sites specifically in the S phase of the cell cycle to promote homologous recombination.

A Translational Model to Improve Early Detection of Epithelial Ovarian Cancers.

Neural network analyses of circulating miRNAs have shown potential as non-invasive screening tests for ovarian cancer. A clinically useful test would detect occult disease when complete cytoreduction is most feasible. Here we used murine xenografts to sensitize a neural network model to detect low volume disease and applied the model to sera from 75 early-stage ovarian cancer cases age-matched to 200 benign adnexal masses or healthy controls. The 14-miRNA model efficiently discriminated tumor bearing animals from controls with 100% sensitivity down to tumor inoculums of 50,000 cells. Among early-stage patient samples, the model performed well with 73% sensitivity at 91% specificity. Applied to a population with 1% disease prevalence, we hypothesize the model would detect most early-stage ovarian cancers while maintaining a negative predictive value of 99.97% (95% CI 99.95%-99.98%). Overall, this supports the concept that miRNAs may be useful as screening markers for early-stage disease.

Combined PARP and HSP90 inhibition: preclinical and Phase 1 evaluation in patients with advanced solid tumours.

PURPOSE: PARP inhibitor resistance may be overcome by combinatorial strategies with agents that disrupt homologous recombination repair (HRR). Multiple HRR pathway components are HSP90 clients, so that HSP90 inhibition leads to abrogation of HRR and sensitisation to PARP inhibition. We performed in vivo preclinical studies of the HSP90 inhibitor onalespib with olaparib and conducted a Phase 1 combination study. PATIENTS AND METHODS: Tolerability and efficacy studies were performed in patient-derived xenograft(PDX) models of ovarian cancer. Clinical safety, tolerability, steady-state pharmacokinetics and preliminary efficacy of olaparib and onalespib were evaluated using a standard 3 + 3 dose-escalation design. RESULTS: Olaparib/onalespib exhibited anti-tumour activity against BRCA1-mutated PDX models with acquired PARPi resistance and PDX models with RB-pathway alterations(CDKN2A loss and CCNE1 overexpression). Phase 1 evaluation revealed that dose levels up to olaparib 300 mg/onalespib 40 mg and olaparib 200 mg/onalespib 80 mg were safe without dose-limiting toxicities. Coadministration of olaparib and onalespib did not appear to affect the steady-state pharmacokinetics of either agent. There were no objective responses, but disease stabilisation ≥24 weeks was observed in 7/22 (32%) evaluable patients including patients with BRCA-mutated ovarian cancers and acquired PARPi resistance and patients with tumours harbouring RB-pathway alterations. CONCLUSIONS: Combining onalespib and olaparib was feasible and demonstrated preliminary evidence of anti-tumour activity.

MicroRNA profiling in a case-control study of African American women with uterine serous carcinoma.

OBJECTIVE: Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer associated with worse survival outcomes in African American (AA) patients. This study evaluated tumor miRNA expression by race, clinical and tumor characteristics, and survival outcomes. METHODS: FFPE tumor tissue from hysterectomy specimens was identified for 29 AA cases. Case matching was performed by computer-based random assignment in a 1:1 ratio with Caucasian controls based on age, stage and histologic subtype (pure vs. mixed). RNA was extracted from 77 specimens (54 tumors and 23 matched normal endometrium). MicroRNA array profiling was performed by microRNA Hi-Power Labeling (Hy3/Hy5) and hybridization to miRCURY LNA microRNA Array 7th Gen. RESULTS: Clinical and treatment characteristics were similar for cases and controls, although use of adjuvant radiation was less common in African Americans (p = 0.03). Of 968 miRNAs analyzed, 649 were differentially expressed in normal endometrium vs. tumor. When compared by race, histologic subtype, stage or presence of LVI, no differentially expressed miRNAs were identified. In patients with disease recurrence at 3 years, the three most upregulated miRNAs were miR-1, miR-21-5p and miR-223. Of these, increased miR-223 expression (>median) was associated with worse OS (p = 0.0496) in an independent dataset (TCGA dataset) comprising of 140 patients with USC (mixed or pure serous). After adjusting for age, ethnicity and BMI, upregulation of miR-223 remained risk factor for death (adjusted HR 2.87, 95% CI 1.00-8.27). CONCLUSIONS: MiRNA profiling did not identify biological differences between AA and Caucasian patients with USC. Upregulation of miR-223 may be a prognostic factor in USC.

A Replication stress biomarker is associated with response to gemcitabine versus combined gemcitabine and ATR inhibitor therapy in ovarian cancer.

In a trial of patients with high grade serous ovarian cancer (HGSOC), addition of the ATR inhibitor berzosertib to gemcitabine improved progression free survival (PFS) compared to gemcitabine alone but biomarkers predictive of treatment are lacking. Here we report a candidate biomarker of response to gemcitabine versus combined gemcitabine and ATR inhibitor therapy in HGSOC ovarian cancer. Patients with replication stress (RS)-high tumors (n = 27), defined as harboring at least one genomic RS alteration related to loss of RB pathway regulation and/or oncogene-induced replication stress achieve significantly prolonged PFS (HR = 0.38, 90% CI, 0.17-0.86) on gemcitabine monotherapy compared to those with tumors without such alterations (defined as RS-low, n = 30). However, addition of berzosertib to gemcitabine benefits only patients with RS-low tumors (gemcitabine/berzosertib HR 0.34, 90% CI, 0.13-0.86) and not patients with RS-high tumors (HR 1.11, 90% CI, 0.47-2.62). Our findings support the notion that the exacerbation of RS by gemcitabine monotherapy is adequate for lethality in RS-high tumors. Conversely, for RS-low tumors addition of berzosertib-mediated ATR inhibition to gemcitabine is necessary for lethality to occur. Independent prospective validation of this biomarker is required.

TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs.

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.