Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new ...therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.
2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of ...5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors,
-oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement.
Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε)-Methylation of ...lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε)-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors.
High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay.
These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The JmjC oxygenases catalyze the N-demethylation of N ε-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified ...assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by mutations in the gene encoding the WAS protein (WASp). Here, induced pluripotent stem cells (iPSCs) were ...derived from a WAS patient (WAS-iPSC) and the endogenous chromosomal WAS locus was targeted with a wtWAS-2A-eGFP transgene using zinc finger nucleases (ZFNs) to generate corrected WAS-iPSC (cWAS-iPSC). WASp and GFP were first expressed in the earliest CD34+CD43+CD45− hematopoietic precursor cells and later in all hematopoietic lineages examined. Whereas differentiation to non-lymphoid lineages was readily obtained from WAS-iPSCs, in vitro T lymphopoiesis from WAS-iPSC was deficient with few CD4+CD8+ double-positive and mature CD3+ T cells obtained. T cell differentiation was restored for cWAS-iPSCs. Similarly, defects in natural killer cell differentiation and function were restored on targeted correction of the WAS locus. These results demonstrate that the defects exhibited by WAS-iPSC-derived lymphoid cells were fully corrected and suggests the potential therapeutic use of gene-corrected WAS-iPSCs.
•Targeted endogenous knockin of Wiskott-Aldrich syndrome (WAS) transgene in WAS-iPSCs•Mutant WAS-iPSCs exhibited defective NK- and T-lymphoid cell development and function•Correction of WAS-iPSCs restored lymphoid cell development and function•These results suggest the potential therapeutic use of gene-corrected WAS-iPSCs
In this article, Davis and colleagues investigated targeted gene correction of induced pluripotent stem cells derived from a patient with Wiskott-Aldrich syndrome (WAS). They employed a knockin strategy in which a WAS transgene construct was selectively integrated into the endogenous WAS locus. Lymphoid cell-specific development and function were selectively defective for WAS-iPSCs and restored for corrected iPSCs.
Abstract
As clinical data are digitized in electronic medical records (EMR), the amount of historical data becomes a challenge for utilization and outcomes studies. An automated approach for ...structuring clinical data into machine-readable format is essential due to scale. We previously developed MMPower, an ElasticSearch-based technology platform for named entity extraction defined by SNOMED conditions, >2,000 Entrez Genes, >900,000 cancer alterations, and ~7,000 FDA-approved and experimental therapeutics/progression that enables clinical trials search. In a pilot study, MMPower was applied to characterize and index oncology EMR for elucidating clinical histories and identifying clinical trial participants. 52,509 individual EMR were collated by Medical Record Number (MRN) across facets including Demographics, Providers/Sites, Diagnosis, Medication, Imaging, Lab results, Pathology, Performance status, and Encounter with Physician Notes. Applying MMPower yielded a total cohort of 43,987 (83.8% of EMR received) EMR with searchable, structured metadata profiles. We found that 39,317 (89.4%) of the total cohort had a cancer diagnosis as defined by SNOMED cancer types. We also extracted an Entrez gene from 36,152 (82.2%) EMR, while cancer alterations or stratification biomarkers such as ER/PR status, ALK fusions, IGH-BCL1, EGFR exon 19 deletion, CDKN2A loss, etc., were identified in 25,208 (57.3%) EMR. Furthermore, we found that the majority of EMR (69.7%) with identifiable genes also contained a cancer alteration. Interestingly, although most EMR (39,615 or 90% of total cohort) held identifiable cancer therapeutics, only 1.7% of the total cohort were flagged as cases of progression. We then determined the feasibility of matching EMR to clinical trials and found that, strikingly, 16.7% of the total cohort were potentially eligible for institution-specific clinical trials. Finally, we sought to enrich individual EMR with molecular alterations data from external lab reports, so we developed a PDF to JSON transform that enables mapping of report results by MRN to our cancer alterations model, yielding a single searchable record from multiple data sources. These results suggest that the ability to structure machine-readable clinical and molecular data for individuals from EMR at scale would accelerate translational research by promoting more efficient clinical trials accrual and could be extended to prevalence calculations, evidence-backed treatment guidance through molecular assertions, biomarkers discovery, and outcomes studies of therapeutic efficacy.
Citation Format: Ryan Duren, Ryan Smith, Nick Tackes, Shane Neeley, James Welsh, Xuan Shirley Li. Scalable assembly of individual patient profiles for clinical trials accrual and research abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3677.
Abstract
Distinguishing genomic events that drive cancer (drivers) from inconsequential damage (passengers) is key to matching patients with biomarker-associated therapies and clinical trials. Whilst ...population level genomic analyses can estimate the proportion of driver events within a given gene, they cannot categorize individual variants. Reliable annotation remains a significant barrier to unlocking the full utility of cancer genomics. GENIE (Genomics Evidence Neoplasia Information Exchange) data was used to generate codon recurrence scores (CRS) for known cancer genes. GENIE was parsed to remove duplicates, sequencing artefact (recurrent variants reported by one institution, invariably associated with amplicon-based sequencing) and hypermutated samples (>15 mutations per megabase). For missense mutations, a codon recurrence of ≥10 was used to classify driver mutations.
The table shows proportion of missense mutations classified as drivers by gene and cancer type, comparing population-based non-synonymous to synonymous mutation ratio; dN/dS (Martincorena 2017, PMID 29056346) with assessment of individual variants by CRS. This comparison demonstrates a high degree of concordance. For a few genes, CRS under called driver mutations (ie VHL in renal cancer) likely due to reduced power with small sample numbers. Importantly, CRS is able to assign driver status to individual missense mutations. Comparison with informatic analyses (PMIDs 28115009, 29247016, 30365005, 31034466) showed equivalent or superior performance of the GENIE approach. These findings demonstrate how a large (and expanding) real-world dataset can be used to predict the driver status of somatic missense mutations at the n=1 variant level. This process is amenable to implementation as a rules-based classification process for somatic missense mutations as part of an automated annotation pipeline.
+corrected dN/dS ratio not significantly different from one. Pan- cancer Breast cancer Renal cancer Lung adenocarcinoma Gene dN/dS CRS dN/dS CRS dN/dS CRS dN/dS CRS BRAF 91% 76% <50% 34% <50% 33% 88% 74% PIK3CA 94% 89% 97% 95% 65%+ 84% 86% 76% KRAS 97% 98% 90% 95% 80%+ 100% 99% 99% IDH1 96% 81% <50% 16% <50% n/a <50% 47% EGFR 52% 51% <50% 7% <50% 17% 88% 81% TP53 96% 96% 99% 97% 82%+ 89% 96% 95% PTEN 94% 54% 90% 59% <50% 47% 70% 32% VHL 80% 22% <50% 8% 99% 33% <50% 0% CDKN2A 50% 51% <50% 43% <50% n/a <50% 46% APC <50% 1% <50% 1% <50% n/a <50% 1% RB1 <50% 6% <50% 6% <50% 50% <50% 5% ARID1A <50% 3% <50% 2% <50% 6% <50% 2% KMT2C <50% 0% <50% 0% <50% n/a <50% 0%
Citation Format: Philip A. Beer, Susanna L. Cooke, Xuan Shirley Li, Andrew V. Biankin. Leveraging the GENIE dataset to distinguish somatic cancer drivers from passenger events in routine oncology practice abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1176.
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