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.

Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity.

Small-molecule inhibitors of PARP1/2, such as olaparib, have been proposed to serve as a synthetic lethal therapy for cancers that harbor BRCA1 or BRCA2 mutations. Indeed, in clinical trials, PARP1/2 inhibitors elicit sustained antitumor responses in patients with germline BRCA gene mutations. In hypothesizing that additional genetic determinants might direct use of these drugs, we conducted a genome-wide synthetic lethal screen for candidate olaparib sensitivity genes. In support of this hypothesis, the set of identified genes included known determinants of olaparib sensitivity, such as BRCA1, RAD51, and Fanconi's anemia susceptibility genes. In addition, the set included genes implicated in established networks of DNA repair, DNA cohesion, and chromatin remodeling, none of which were known previously to confer sensitivity to PARP1/2 inhibition. Notably, integration of the list of candidate sensitivity genes with data from tumor DNA sequencing studies identified CDK12 deficiency as a clinically relevant biomarker of PARP1/2 inhibitor sensitivity. In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination, and reduced expression of BRCA1. As one of only nine genes known to be significantly mutated in HGS-OVCa, CDK12 has properties that should confirm interest in its use as a biomarker, particularly in ongoing clinical trials of PARP1/2 inhibitors and other agents that trigger replication fork arrest.

BMN 673, a novel and highly potent PARP1/2 inhibitor for the treatment of human cancers with DNA repair deficiency.

PURPOSE: PARP1/2 inhibitors are a class of anticancer agents that target tumor-specific defects in DNA repair. Here, we describe BMN 673, a novel, highly potent PARP1/2 inhibitor with favorable metabolic stability, oral bioavailability, and pharmacokinetic properties. EXPERIMENTAL DESIGN: Potency and selectivity of BMN 673 was determined by biochemical assays. Anticancer activity either as a single-agent or in combination with other antitumor agents was evaluated both in vitro and in xenograft cancer models. RESULTS: BMN 673 is a potent PARP1/2 inhibitor (PARP1 IC50 = 0.57 nmol/L), but it does not inhibit other enzymes that we have tested. BMN 673 exhibits selective antitumor cytotoxicity and elicits DNA repair biomarkers at much lower concentrations than earlier generation PARP1/2 inhibitors (such as olaparib, rucaparib, and veliparib). In vitro, BMN 673 selectively targeted tumor cells with BRCA1, BRCA2, or PTEN gene defects with 20- to more than 200-fold greater potency than existing PARP1/2 inhibitors. BMN 673 is readily orally bioavailable, with more than 40% absolute oral bioavailability in rats when dosed in carboxylmethyl cellulose. Oral administration of BMN 673 elicited remarkable antitumor activity in vivo; xenografted tumors that carry defects in DNA repair due to BRCA mutations or PTEN deficiency were profoundly sensitive to oral BMN 673 treatment at well-tolerated doses in mice. Synergistic or additive antitumor effects were also found when BMN 673 was combined with temozolomide, SN38, or platinum drugs. CONCLUSION: BMN 673 is currently in early-phase clinical development and represents a promising PARP1/2 inhibitor with potentially advantageous features in its drug class.

A genetic screen using the PiggyBac transposon in haploid cells identifies Parp1 as a mediator of olaparib toxicity.

Genetic perturbation screens have the potential to dissect a wide range of cellular phenotypes. Such screens have historically been difficult in diploid mammalian cells. The recent derivation of haploid embryonic stem cells provides an opportunity to cause loss of function mutants with a random mutagen in a mammalian cell with a normal genetic background. We describe an approach to genetic screens that exploits the highly active piggyBac transposon in haploid mammalian cells. As an example of haploid transposon (HTP) screening, we apply this approach to identifying determinants of cancer drug toxicity and resistance. In a screen for 6-thioguanine resistance we recovered components of the DNA mismatch repair pathway, a known requirement for toxicity. In a further screen for resistance to the clinical poly(ADP-ribose) polymerase (PARP) inhibitor olaparib we recovered multiple Parp1 mutants. Our results show that olaparib toxicity to normal cells is mediated predominantly via Parp1, and suggest that the clinical side effects of olaparib may be on target. The transposon mutant libraries are stable and can be readily reused to screen other drugs. The screening protocol described has several advantages over other methods such as RNA interference: it is rapid and low cost, and mutations can be easily reverted to establish causality.

Sequence based analysis of U-2973, a cell line established from a double-hit B-cell lymphoma with concurrent MYC and BCL2 rearrangements.

BACKGROUND: Double-hit lymphoma is a complex and highly aggressive sub-type of B-cell lymphoma, which has recently been classified and is an area of active research interest due to the poor prognosis for patients with this disease. It is characterized by the presence of both an activating MYC chromosomal translocation and a simultaneous additional oncogenic translocation, often of the BCL2 gene. Recently, a cell line was established from a patient with this complex lymphoma and analyzed using conventional tools revealing it contains both MYC and BCL2 translocation events. FINDINGS: In this work, we reanalyzed the genome of the cell line using next generation whole genome sequencing technology in order to catalogue translocations, insertions and deletions which may contribute to the pathology of this lymphoma type. CONCLUSIONS: We describe the cell line in much greater detail, and pinpoint the exact locations of the chromosomal breakpoints. We also find several rearrangements within cancer-associated genes, which were not found using conventional tools, suggesting that high throughput sequencing may reveal novel targets for therapy, which could be used concurrently with existing treatments.

The promise of combining inhibition of PI3K and PARP as cancer therapy.

Analyses of in vitro and patient-derived in vivo models of breast cancer reveal that a combination of inhibitors of the enzymes PARP and phosphoinositide 3-kinase (PI3K) is a potentially effective treatment regimen for breast cancer tumors with elevated activity of the PI3K pathway.

Synthetic lethal approaches to breast cancer therapy.

The promise of personalized therapy for breast cancer is that therapeutic efficacy will be increased while toxic effects are reduced to a minimum. To achieve this goal, there is now an emphasis on the design of therapies that are based not only on the clinical manifestations of the disease, but also on the underlying molecular and cellular biology of cancer. However, identifying targets for personalized therapies in breast cancer is challenging. Here, we describe how biological concepts such as synthetic lethality and oncogene addiction can be used to identify new therapeutic targets and approaches. We discuss the current clinical developments in implementing synthetic lethality therapies, and highlight new ways in which this approach could be used to target specific subsets of breast cancer.