Helen N. Pemberton

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, Fanconis 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.).

Breast cancer intratumor genetic heterogeneity: causes and implications

There is burgeoning evidence to suggest that tumor evolution follows the laws of Darwinian evolution, whereby individual tumor cell clones harbor private genetic aberrations in addition to the founder mutations, and that these distinct populations of cancer cells interact in competitive and mutualistic manners. The combined effect of genetic and epigenetic instability, and differential selective pressures according to the microenvironment and therapeutic interventions, create many different evolutionary routes such that intratumor heterogeneity is inevitable. Numerous cytogenetic, comparative genomic hybridization and, more recently, massively parallel sequencing studies have generated indisputable evidence of this phenomenon. The impact of intratumor heterogeneity on response and resistance to therapy is beginning to be understood; this information may prove crucial for the potentials of personalized medicine to be realized. In this review, the evidence of intratumor heterogeneity in breast cancer, its potential causes and implications for the clinical management of breast cancer patients are discussed.

ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A

Identifying genetic biomarkers of synthetic lethal drug sensitivity effects provides one approach to the development of targeted cancer therapies. Mutations in ARID1A represent one of the most common molecular alterations in human cancer, but therapeutic approaches that target these defects are not yet clinically available. We demonstrate that defects in ARID1A sensitize tumour cells to clinical inhibitors of the DNA damage checkpoint kinase, ATR, both in vitro and in vivo. Mechanistically, ARID1A deficiency results in topoisomerase 2A and cell cycle defects, which cause an increased reliance on ATR checkpoint activity. In ARID1A mutant tumour cells, inhibition of ATR triggers premature mitotic entry, genomic instability and apoptosis. The data presented here provide the pre-clinical and mechanistic rationale for assessing ARID1A defects as a biomarker of single-agent ATR inhibitor response and represents a novel synthetic lethal approach to targeting tumour cells.

Large-Scale Profiling of Kinase Dependencies in Cancer Cell Lines

Summary One approach to identifying cancer-specific vulnerabilities and therapeutic targets is to profile genetic dependencies in cancer cell lines. Here, we describe data from a series of siRNA screens that identify the kinase genetic dependencies in 117 cancer cell lines from ten cancer types. By integrating the siRNA screen data with molecular profiling data, including exome sequencing data, we show how vulnerabilities/genetic dependencies that are associated with mutations in specific cancer driver genes can be identified. By integrating additional data sets into this analysis, including protein-protein interaction data, we also demonstrate that the genetic dependencies associated with many cancer driver genes form dense connections on functional interaction networks. We demonstrate the utility of this resource by using it to predict the drug sensitivity of genetically or histologically defined subsets of tumor cell lines, including an increased sensitivity of osteosarcoma cell lines to FGFR inhibitors and SMAD4 mutant tumor cells to mitotic inhibitors.

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1–S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472–84. ©2016 AACR.

Modeling Therapy Resistance in BRCA1/2-Mutant Cancers

Although PARP inhibitors target BRCA1- or BRCA2-mutant tumor cells, drug resistance is a problem. PARP inhibitor resistance is sometimes associated with the presence of secondary or “revertant” mutations in BRCA1 or BRCA2. Whether secondary mutant tumor cells are selected for in a Darwinian fashion by treatment is unclear. Furthermore, how PARP inhibitor resistance might be therapeutically targeted is also poorly understood. Using CRISPR mutagenesis, we generated isogenic tumor cell models with secondary BRCA1 or BRCA2 mutations. Using these in heterogeneous in vitro culture or in vivo xenograft experiments in which the clonal composition of tumor cell populations in response to therapy was monitored, we established that PARP inhibitor or platinum salt exposure selects for secondary mutant clones in a Darwinian fashion, with the periodicity of PARP inhibitor administration and the pretreatment frequency of secondary mutant tumor cells influencing the eventual clonal composition of the tumor cell population. In xenograft studies, the presence of secondary mutant cells in tumors impaired the therapeutic effect of a clinical PARP inhibitor. However, we found that both PARP inhibitor–sensitive and PARP inhibitor–resistant BRCA2 mutant tumor cells were sensitive to AZD-1775, a WEE1 kinase inhibitor. In mice carrying heterogeneous tumors, AZD-1775 delivered a greater therapeutic benefit than olaparib treatment. This suggests that despite the restoration of some BRCA1 or BRCA2 gene function in “revertant” tumor cells, vulnerabilities still exist that could be therapeutically exploited. Mol Cancer Ther; 16(9); 2022–34. ©2017 AACR.

ATR Is a Therapeutic Target in Synovial Sarcoma

Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by expression of SS18-SSX fusions, where treatment options are limited. To identify therapeutically actionable genetic dependencies in SS, we performed a series of parallel, high-throughput small interfering RNA (siRNA) screens and compared genetic dependencies in SS tumor cells with those in >130 non-SS tumor cell lines. This approach revealed a reliance of SS tumor cells upon the DNA damage response serine/threonine protein kinase ATR. Clinical ATR inhibitors (ATRi) elicited a synthetic lethal effect in SS tumor cells and impaired growth of SS patient-derived xenografts. Oncogenic SS18-SSX family fusion genes are known to alter the composition of the BAF chromatin-remodeling complex, causing ejection and degradation of wild-type SS18 and the tumor suppressor SMARCB1. Expression of oncogenic SS18-SSX fusion proteins caused profound ATRi sensitivity and a reduction in SS18 and SMARCB1 protein levels, but an SSX18-SSX1 Δ71-78 fusion containing a C-terminal deletion did not. ATRi sensitivity in SS was characterized by an increase in biomarkers of replication fork stress (increased γH2AX, decreased replication fork speed, and increased R-loops), an apoptotic response, and a dependence upon cyclin E expression. Combinations of cisplatin or PARP inhibitors enhanced the antitumor cell effect of ATRi, suggesting that either single-agent ATRi or combination therapy involving ATRi might be further assessed as candidate approaches for SS treatment. Cancer Res; 77(24); 7014-26. ©2017 AACR.

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.

Genome-wide barcoded transposon screen for cancer drug sensitivity in haploid mouse embryonic stem cells

We describe a screen for cellular response to drugs that makes use of haploid embryonic stem cells. We generated ten libraries of mutants with piggyBac gene trap transposon integrations, totalling approximately 100,000 mutant clones. Random barcode sequences were inserted into the transposon vector to allow the number of cells bearing each insertion to be measured by amplifying and sequencing the barcodes. These barcodes were associated with their integration sites by inverse PCR. We exposed these libraries to commonly used cancer drugs and profiled changes in barcode abundance by Ion Torrent sequencing in order to identify mutations that conferred sensitivity. Drugs tested included conventional chemotherapeutics as well as targeted inhibitors of topoisomerases, poly(ADP-ribose) polymerase (PARP), Hsp90 and WEE1.

Functional diversity and cooperativity between subclonal populations of pediatric glioblastoma and diffuse intrinsic pontine glioma cells

The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments.Genomic and functional analysis of intratumor heterogeneity in pediatric glioma uncovers early clonal divergence and stable spontaneous cooperation between subclonal populations throughout tumor evolution.