DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer.

BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib. METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies. RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib. CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).

Elevated APOBEC3B expression drives a kataegic-like mutation signature and replication stress-related therapeutic vulnerabilities in p53-defective cells.

BACKGROUND: Elevated APOBEC3B expression in tumours correlates with a kataegic pattern of localised hypermutation. We assessed the cellular phenotypes associated with high-level APOBEC3B expression and the influence of p53 status on these phenotypes using an isogenic system. METHODS: We used RNA interference of p53 in cells with inducible APOBEC3B and assessed DNA damage response (DDR) biomarkers. The mutational effects of APOBEC3B were assessed using whole-genome sequencing. In vitro small-molecule inhibitor sensitivity profiling was used to identify candidate therapeutic vulnerabilities. RESULTS: Although APOBEC3B expression increased the incorporation of genomic uracil, invoked DDR biomarkers and caused cell cycle arrest, inactivation of p53 circumvented APOBEC3B-induced cell cycle arrest without reversing the increase in genomic uracil or DDR biomarkers. The continued expression of APOBEC3B in p53-defective cells not only caused a kataegic mutational signature but also caused hypersensitivity to small-molecule DDR inhibitors (ATR, CHEK1, CHEK2, PARP, WEE1 inhibitors) as well as cisplatin/ATR inhibitor and ATR/PARP inhibitor combinations. CONCLUSIONS: Although loss of p53 might allow tumour cells to tolerate elevated APOBEC3B expression, continued expression of this enzyme might impart a number of therapeutic vulnerabilities upon tumour cells.

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

Functional screening reveals HORMAD1-driven gene dependencies associated with translesion synthesis and replication stress tolerance.

HORMAD1 expression is usually restricted to germline cells, but it becomes mis-expressed in epithelial cells in ~60% of triple-negative breast cancers (TNBCs), where it is associated with elevated genomic instability (1). HORMAD1 expression in TNBC is bimodal with HORMAD1-positive TNBC representing a biologically distinct disease group. Identification of HORMAD1-driven genetic dependencies may uncover novel therapies for this disease group. To study HORMAD1-driven genetic dependencies, we generated a SUM159 cell line model with doxycycline-inducible HORMAD1 that replicated genomic instability phenotypes seen in HORMAD1-positive TNBC (1). Using small interfering RNA screens, we identified candidate genes whose depletion selectively inhibited the cellular growth of HORMAD1-expressing cells. We validated five genes (ATR, BRIP1, POLH, TDP1 and XRCC1), depletion of which led to reduced cellular growth or clonogenic survival in cells expressing HORMAD1. In addition to the translesion synthesis (TLS) polymerase POLH, we identified a HORMAD1-driven dependency upon additional TLS polymerases, namely POLK, REV1, REV3L and REV7. Our data confirms that out-of-context somatic expression of HORMAD1 can lead to genomic instability and reveals that HORMAD1 expression induces dependencies upon replication stress tolerance pathways, such as translesion synthesis. Our data also suggest that HORMAD1 expression could be a patient selection biomarker for agents targeting replication stress.

A Flexible, Pooled CRISPR Library for Drug Development Screens.

Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. However, some experimental paradigms prove especially challenging and require carefully and appropriately adapted screening approaches. In particular, negative selection (or sensitivity) screening, often the most experimentally desirable modality of screening, has remained a challenge in drug discovery. Here we assess whether our new, modular genome-wide pooled CRISPR library can improve negative selection CRISPR screening and add utility throughout the drug development pipeline. Our pooled library is split into three parts, allowing it to be scaled to accommodate the experimental challenges encountered during drug development, such as target identification using unlimited cell numbers compared with target identification studies for cell populations where cell numbers are limiting. To test our new library, we chose to look for drug-gene interactions using a well-described small molecule inhibitor targeting poly(ADP-ribose) polymerase 1 (PARP1), and in particular to identify genes which sensitise cells to this drug. We simulate hit identification and performance using each library partition and support these findings through orthogonal drug combination cell panel screening. We also compare our data with a recently published CRISPR sensitivity dataset obtained using the same PARP1 inhibitor. Overall, our data indicate that generating a comprehensive CRISPR knockout screening library where the number of guides can be scaled to suit the biological question being addressed allows a library to have multiple uses throughout the drug development pipeline, and that initial validation of hits can be achieved through high-throughput cell panels screens where clinical grade chemical or biological matter exist.

Therapeutic vulnerabilities in the DNA damage response for the treatment of ATRX mutant neuroblastoma.

BACKGROUND: In neuroblastoma, genetic alterations in ATRX, define a distinct poor outcome patient subgroup. Despite the need for new therapies, there is a lack of available models and a dearth of pre-clinical research. METHODS: To evaluate the impact of ATRX loss of function (LoF) in neuroblastoma, we utilized CRISPR-Cas9 gene editing to generate neuroblastoma cell lines isogenic for ATRX. We used these and other models to identify therapeutically exploitable synthetic lethal vulnerabilities associated with ATRX LoF. FINDINGS: In isogenic cell lines, we found that ATRX inactivation results in increased DNA damage, homologous recombination repair (HRR) defects and impaired replication fork processivity. In keeping with this, high-throughput compound screening showed selective sensitivity in ATRX mutant cells to multiple PARP inhibitors and the ATM inhibitor KU60019. ATRX mutant cells also showed selective sensitivity to the DNA damaging agents, sapacitabine and irinotecan. HRR deficiency was also seen in the ATRX deleted CHLA-90 cell line, and significant sensitivity demonstrated to olaparib/irinotecan combination therapy in all ATRX LoF models. In-vivo sensitivity to olaparib/irinotecan was seen in ATRX mutant but not wild-type xenografts. Finally, sustained responses to olaparib/irinotecan therapy were seen in an ATRX deleted neuroblastoma patient derived xenograft. INTERPRETATION: ATRX LoF results in specific DNA damage repair defects that can be therapeutically exploited. In ATRX LoF models, preclinical sensitivity is demonstrated to olaparib and irinotecan, a combination that can be rapidly translated into the clinic. FUNDING: This work was supported by Christopher's Smile, Neuroblastoma UK, Cancer Research UK, and the Royal Marsden Hospital NIHR BRC.

Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance.

Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.

E-Cadherin/ROS1 Inhibitor Synthetic Lethality in Breast Cancer.

The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin-defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin-defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin-defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498-515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.

Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness.

Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS.

Author Correction: Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.