We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment ...results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.
The short‐chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes, most of which are NAD(H)‐dependent or NADP(H)‐dependent oxidoreductases. They display a wide substrate ...spectrum, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics. On the basis of characteristic sequence motifs, the SDRs are subdivided into two main (classical and extended) and three smaller (divergent, intermediate, and complex) families. Despite low residue identities in pairwise comparisons, the three‐dimensional structure among the SDRs is conserved and shows a typical Rossmann fold. Here, we used a bioinformatics approach to determine whether and which SDRs are present in cyanobacteria, microorganisms that played an important role in our ecosystem as the first oxygen producers. Cyanobacterial SDRs could indeed be identified, and were clustered according to the SDR classification system. Furthermore, because of the early availability of its genome sequence and the easy application of transformation methods, Synechocystis sp. PCC 6803, one of the most important cyanobacterial strains, was chosen as the model organism for this phylum. Synechocystis sp. SDRs were further analysed with bioinformatics tools, such as hidden Markov models (HMMs). It became evident that several cyanobacterial SDRs show remarkable sequence identities with SDRs in other organisms. These so‐called ‘homologous’ proteins exist in plants, model organisms such as Drosophila melanogaster and Caenorhabditis elegans, and even in humans. As sequence identities of up to 60% were found between Synechocystis and humans, it was concluded that SDRs seemed to have been well conserved during evolution, even after dramatic terrestrial changes such as the conversion of the early reducing atmosphere to an oxidizing one by cyanobacteria.
Short‐chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes with a wide substrate spectrum. This bioinformatics approach validated the presence of SDRs in cyanobacteria, the first oxygen producers. It showed sequence identities of up to 60% to homologous proteins in other organisms, remarkably also in human, indicating that SDR proteins are highly conserved during evolution, even after dramatic terrestrial changes.
GPX4 represents a promising yet difficult-to-drug therapeutic target for the treatment of, among others, drug-resistant cancers. Although most GPX4 inhibitors rely on a chloroacetamide moiety to ...modify covalently the protein’s catalytic selenocysteine residue, the discovery and mechanistic elucidation of structurally diverse GPX4-inhibiting molecules have uncovered novel electrophilic warheads that bind and inhibit GPX4. Here, we report our discovery that diacylfuroxans can act as masked nitrile oxide prodrugs that inhibit GPX4 covalently with unique cellular and biochemical reactivity compared to existing classes of GPX4 inhibitors. These observations illuminate a novel molecular mechanism of action for biologically active furoxans and also expand the collection of reactive groups capable of targeting GPX4.
Abstract
BACKGROUND
Tumors involving the central nervous system (CNS) include over 200 primary and metastatic subtypes with major clinical impact. Research on CNS neoplasms has been hampered by the ...lack of appropriate models for many subtypes. We established a robust workflow and systematic culturing approach to create cancer cell line models from all adult and pediatric CNS cancer patients and here report the results of these ongoing efforts.
METHODS
Tumor samples from consented patients with CNS cancers were systematically collected from 2008–18. Tumors were grown in different media and substrates and growth verified by >5 passages. Genomic verification was performed using NGS analysis (focused SNV and CNA) and expression profiling assays.
RESULTS
We attempted to generate cell line models from >1500 consented brain tumor patients at the Brigham and Women’s and Boston Children’s Hospital (IRB-10–417) under the DFCI Living Tissue Bank Program as well as within the Broad Institute Cancer Cell Line Factory (CCLF) Project and the Leuven Living Tissue Bank (IRB-S59804, Belgium). 120 different tumor types, including high and low-grade brain tumors of both adult and pediatric origin, were evaluated for in vitro growth in >2000 culturing attempts. The success rate of growing high-grade tumors (i.e. glioma and of other origin) was robustly high (~50%), while the growth verification rate for low-grade tumors remained low (<1%). Overall, growth beyond passage 5 was achieved in ~30% of cases, and of the growth verified models, also ~30% were genomically verified to represent cancer, with the majority maintaining genomic and/or transcriptional features. In addition to primary CNS tumors, we were now also able to grow metastatic cultures, resulting in >150 novel model systems covering >20 primary and metastatic diagnoses.
CONCLUSION
Patient-derived cell lines may be created at scale from primary and metastatic CNS tumors to support pre-clinical cancer research but technological improvements will be required to culture even more tumor types.
A major therapeutic challenge in musculoskeletal regenerative medicine is how to effectively replenish bone tissue lost due to pathological conditions such as fracture, osteoporosis, or rheumatoid ...arthritis. Mesenchymal stem cells are currently investigated for applications in bone-tissue engineering and human bone marrow-derived mesenchymal stem cells (hMSCs) could be a promising source for generation of tissue-engineered bone. However, the therapeutic potential of MSCs has not been fully exploited due to a lack of knowledge regarding the identity, nature, and differentiation of hMSCs. Epigenetic mechanisms regulating the chromatin structure as well as specific gene transcription are crucial in determination of stem cell differentiation. With the aim to systematically identify epigenetic factors that modulate MSC differentiation, the work in this thesis encompasses an approach to identify epigenetic mechanisms underlying, initiating, and promoting osteoblast differentiation, and the investigation of individual epigenetic modulators. Various osteogenic inducers were validated for differentiation of MSCs and an assay allowing assessment of differentiation outcome was developed. This assay was subsequently employed in knockdown experiments with lentiviral short hairpin RNAs and inhibitor screens with small molecules targeting putative druggable epigenetic modulator classes. This approach identified around 100 epigenetic modulator candidates involved in osteoblast differentiation, of these candidates approximately 2/3 downregulated and 1/3 upregulated alkaline phosphatase (ALP) activity. Serving as a proof-of-concept, orthogonal validation experiments employing locked nucleic acid (LNA) knockdown were performed to validate a subset of candidates. Two identified target genes were selected for further investigation. Bromodomain-containing protein 4 (BRD4) was identified as one component of epigenetic regulation; its inhibition led to a decrease in ALP expression, downregulation of key osteoblast transcription factors Runx2 and Osterix, as well as impaired bone matrix formation. Knockdown of lysine (K)-specific demethylase 1A (KDM1A/LSD1) upregulated ALP activity and treatment with a small molecule inhibitor targeting KDM1A led to an increase in ALP, RUNX2, and bone sialoprotein expression. Intriguingly, in a transgenic mouse model overexpressing Kdm1a a decrease in bone volume and bone mineral density was observed, thus supporting the hypothesis that KDM1A is a central regulator of osteoblast differentiation.
A major therapeutic challenge in musculoskeletal regenerative medicine is how to effectively replenish bone tissue lost due to pathological conditions such as fracture, osteoporosis, or rheumatoid ...arthritis. Mesenchymal stem cells are currently investigated for applications in bone-tissue engineering and human bone marrow-derived mesenchymal stem cells (hMSCs) could be a promising source for generation of tissue-engineered bone. However, the therapeutic potential of MSCs has not been fully exploited due to a lack of knowledge regarding the identity, nature, and differentiation of hMSCs. Epigenetic mechanisms regulating the chromatin structure as well as specific gene transcription are crucial in determination of stem cell differentiation. With the aim to systematically identify epigenetic factors that modulate MSC differentiation, the work in this thesis encompasses an approach to identify epigenetic mechanisms underlying, initiating, and promoting osteoblast differentiation, and the investigation of individual epigenetic modulators. Various osteogenic inducers were validated for differentiation of MSCs and an assay allowing assessment of differentiation outcome was developed. This assay was subsequently employed in knockdown experiments with lentiviral short hairpin RNAs and inhibitor screens with small molecules targeting putative druggable epigenetic modulator classes. This approach identified around 100 epigenetic modulator candidates involved in osteoblast differentiation, of these candidates approximately 2/3 downregulated and 1/3 upregulated alkaline phosphatase (ALP) activity. Serving as a proof-of-concept, orthogonal validation experiments employing locked nucleic acid (LNA) knockdown were performed to validate a subset of candidates. Two identified target genes were selected for further investigation. Bromodomain-containing protein 4 (BRD4) was identified as one component of epigenetic regulation; its inhibition led to a decrease in ALP expression, downregulation of key osteoblast transcription factors Runx2 and Osterix, as well as impaired bone matrix formation. Knockdown of lysine (K)-specific demethylase 1A (KDM1A/LSD1) upregulated ALP activity and treatment with a small molecule inhibitor targeting KDM1A led to an increase in ALP, RUNX2, and bone sialoprotein expression. Intriguingly, in a transgenic mouse model overexpressing Kdm1a a decrease in bone volume and bone mineral density was observed, thus supporting the hypothesis that KDM1A is a central regulator of osteoblast differentiation.
Increased global connectivity has catalyzed technological development in almost all industries, in part through the facilitation of novel collaborative structures. Notably, open innovation and ...crowd-sourcing-of expertise and/or funding-has tremendous potential to increase the efficiency with which biomedical ecosystems interact to deliver safe, efficacious and affordable therapies to patients. Consequently, such practices offer tremendous potential in advancing development of cellular therapies. In this vein, the CASMI Translational Stem Cell Consortium (CTSCC) was formed to unite global thought-leaders, producing academically rigorous and commercially practicable solutions to a range of challenges in pluripotent stem cell translation. Critically, the CTSCC research agenda is defined through continuous consultation with its international funding and research partners. Herein, initial findings for all research focus areas are presented to inform global product development strategies, and to stimulate continued industry interaction around biomanufacturing, strategic partnerships, standards, regulation and intellectual property and clinical adoption.
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