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 ARID1Amutant OCCC. Mol Cancer Ther; 15(7); 1472-84. Ó2016 AACR.</p

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

The Toilet Taboo: The Effectiveness of Sanitation Projects in India

Despite India’s advancements in development and industry, it is also the nation with the greatest prevalence of open defecation in the world. This paper examines India as a case study in order to understand some of the barriers to sanitation projects. Specifically, these barriers include: top-down governmental approaches such as inconsistent and ill targeted funding, subsidizing, poor quality toilets when built, and incentivizing. In addition, there is a need to transition to Community-Led Total Sanitation models. This can be done by improving health awareness and integrating vulnerable populations in sanitation campaigns. Additional recommendations for future projects, including further use of technology as seen in the current Clean India Mission, are also made throughout this paper. These findings can provide useful methods to improve sanitation in India, which not only has applications to current sanitation policies in the country, but could also serve as a model to benefit other countries facing similar concerns

Primary pulmonary synovial sarcoma: Diagnosis on squash smears

Synovial sarcomas are rare tumors accounting for approximately 5-10% of soft tissue sarcomas. They occur predominantly in the extremities, followed by head and neck. Primary pulmonary sarcomas are very rare and comprise only 0.5% of all primary lung malignancies. The diagnosis is established only after sarcomas like primary lung malignancies, and metastatic sarcomas have been excluded. For synovial sarcomas that arise at unusual locations, a definitive diagnosis is challenging and requires the use of ancillary diagnostic procedures such as immunohistochemistry (IHC) and molecular genetic techniques for confirmation of diagnosis. We report a case of 29-year-old male who had right lower lobe lung mass. He underwent right lower lobectomy. Intraoperative squash smears revealed spindle cell sarcoma. Subsequent histopathology and IHC confirmed the diagnosis as synovial sarcoma. We report this case on account of its rarity and to emphasize the utility of intraoperative squash smears in the diagnosis of such cases, which has been under-utilized in clinical practice

Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function

Parkin is an ubiquitin-protein ligase (E3), mutations of which cause juvenile onset – autosomal recessive Parkinson's disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2-DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (±2-fold change; p<0.05) using 2-DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2-DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells

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

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

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.Health Research BoardScience Foundation IrelandWellcome TrustBreast Cancer NowCancer Research UKVertex PharmaceuticalsNational Health Servic