Biology subjects

Garrett, M. D.

Publications and source records attributed to Garrett, M. D..

7 recordsLinked to original sources

bioRxiv2025

Reduced 4E-BP1 activity leads to acquired capivasertib resistance via increased cap-dependent protein synthesis.

BackgroundThe PI3K/AKT/mTOR (PAM) pathway is frequently dysregulated in human cancers and is a major target of therapeutic intervention, exemplified by the newly approved AKT inhibitor capivasertib. However, clinical responses are often limited by the emergence of acquired drug resistance. MethodsTo uncover potential mechanisms underlying acquired resistance to capivasertib, we developed a drug-resistant ovarian cancer model (A2780 254R) through chronic exposure to the capivasertib precursor CCT129254. ResultsThe 254R cells exhibit broad cross-resistance to AKT inhibitors, including capivasertib, along with mTORC1 and PI3K inhibitors, in the absence of AKT mutations or elevated kinase activity. A defining feature of resistance is the reduced expression and phosphorylation of 4E-BP1, a key negative regulator of cap-dependent protein synthesis (CDPS). mcGTP pull-down assays revealed impaired 4E-BP1-eIF4E binding and persistent eIF4F complex formation, which was consistent with enhanced CDPS. An [~]9-fold increase in CDPS activity was observed in 254R cells, without corresponding increases in global protein synthesis or the expression of canonical eIF4E targets. Strikingly, ectopic expression of wild-type or constitutively active 4E-BP1 restored capivasertib sensitivity, re-established the 4E-BP1-eIF4E interaction, and significantly reduced resistance to capivasertib. These findings identify impaired 4E-BP1 function and elevated CDPS as central mediators of acquired resistance to capivasertib. ConclusionOverall, this work underscores the therapeutic vulnerability of the translational machinery to AKT inhibitor resistance. Targeting the eIF4F complex may offer a promising strategy to overcome acquired resistance, and the expression or phosphorylation of 4E-BP1 and eIF4E may serve as predictive biomarkers to guide use of capivasertib in the clinic.

cancer biology↗

bioRxiv2025

Identification of molecular pathways involved in the anti-liver cancer activity of madecassic acid

Natural products are a time-tested source of medicinal compounds, and there is huge interest in discovery of new drugs from plant sources. However, in many cases the effects are not as well understood, strong, or selective, as would be hoped. An example of this is madecassic acid (MA), which has promising activity against liver cancer, activity which can be improved through chemical modifications, although a molecular understanding of its mechanism of action is unknown. In this report we have used chemical proteomics to identify the proteins with which madecassic interacts in liver cancer cells, and used RNAseq to support those findings, as well as showing more precisely how chemical modifications can focus and amplify MA activity. Our results show that madecassic acid interacts with a number of proteins relating to metabolism, nucleic acid processing, and protein folding, which have been previously identified as linked to liver cancer. This provides a route from phenotypic- to target based-drug discovery and develop new potential treatments for a globally challenging disease.

pharmacology and toxicology↗

bioRxiv2025

Regioselectively Acetylated Silybin Derivatives and their Cytotoxic Activity in Liver Cancer Cells

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=48 SRC="FIGDIR/small/638106v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@13744c8org.highwire.dtl.DTLVardef@184bb9borg.highwire.dtl.DTLVardef@138f442org.highwire.dtl.DTLVardef@2c45ca_HPS_FORMAT_FIGEXP M_FIG C_FIG Natural products continue to serve as a valuable source of starting points for therapeutic agents. Six silybin derivatives have been synthesized and tested in vitro for their cytotoxic effect on the human hepatocellular carcinoma line HepG2 using the MTT assay. Results indicate that the 3,5,7,20,23-penta-O-acetyl-2,3-dehydrosilybin possesses a stronger cytotoxic effect than that of the parent silybin by a factor of 12. This compound was further evaluated for its cytotoxic activity on Hep3B, Huh7, and Huh7R hepatocellular carcinoma cell lines, giving similar potencies, arresting the cell cycle at S phase and causing apoptosis. These results illustrate the potential of silybin derivatives as therapeutics against liver cancer.

pharmacology and toxicology↗

bioRxiv2024

Phenotypic plasticity in a novel set of EGFR tyrosine kinase inhibitor-adapted non-small cell lung cancer cell lines

Here, we introduce sublines of the EGFR-mutant non-small cell lung cancer (NSCLC) cell lines HCC827 and HCC4006 adapted to the EGFR kinase inhibitors gefitinib (HCC827rGEFI2{micro}M, HCC4006rGEFI1{micro}M), erlotinib (HCC827rERLO2{micro}M, HCC4006rERLO1{micro}M), and afatinib (HCC827rAFA50nM, HCC4006rAFA100nM). All sublines displayed resistance to gefitinib, erlotinib, afatinib, and the third-generation EGFR kinase inhibitor osimertinib that overcomes T790M-mediated resistance. HCC4006rERLO1{micro}M displayed a spindle-like morphology in agreement with previous findings that had detected epithelial-mesenchymal-transition (EMT) in its precursor cell line HCC4006rERLO0.5{micro}M. EMT had also been reported for the HCC4006rGEFI1{micro}M precursor cell line HCC4006rGEFI0.5{micro}M and for HCC4006rAFA100nM, but the morphologies of HCC4006rGEFI1{micro}M or HCC4006rAFA100nM did not support this, suggesting plasticity in EMT regulation during the drug adaptation process and in established resistant cell lines. Accordingly, HCC4006rERLO1{micro}M displayed resistance to MEK and AKT inhibitors in contrast to its precursor HCC4006rERLO0.5{micro}M. We also detected metabolic plasticity, i.e., a temporary Warburg metabolism, in HCC4006 and HCC827rGEFI2{micro}M. Response profiles to cytotoxic anti-cancer drugs, kinase inhibitors, and HDAC inhibitors resulted in complex patterns that were specific for each individual subline without obvious overlaps, indicating individual resistance phenotypes. All resistant sublines remained sensitive or displayed collateral sensitivity to at least one of the investigated drugs. In conclusion, the comparison of EGFR kinase-resistant NSCLC sublines with their precursor cell lines that had been previously characterised at a lower resistance level and metabolic investigations indicated phenotypic plasticity during the resistance formation process and in established cell lines. This plasticity may contribute to the well-known variability in cell line phenotypes observed between different laboratories and in intra-laboratory experiments.

cancer biology↗

bioRxiv2024

Sequence-defined oligophosphoesters for selective inhibition of the KRAS G12D/RAF1 interaction

Rat Sarcoma (RAS) genes are the most frequently mutated genes in cancer, with KRAS being the most predominant oncogene, yet they have proved extremely difficult to drug because they operate primarily through protein-protein interactions (PPIs) which lack an obvious pocket for small molecules. Sequence-defined synthetic oligomers could combine the precision and customisability of synthetic molecules with the size requirements to address entire protein-protein interaction surfaces. We have adapted the phosphoramidite chemistry of oligonucleotide synthesis to produce a library of nearly one million non-nucleosidic oligophosphoester sequences - phosphoestamers - and used a fluorescent-activated bead sorting (FABS) process to select oligomers that inhibit the interaction between KRASG12D (the most prevalent, and undrugged, mutant) and RAF, a downstream effector of RAS whose activation results in cell proliferation. Hits were identified using tandem mass spectrometry, and validation showed effective inhibition with IC50 values as low as 25 nM, and excellent selectivity for the mutant over the wild type form. These findings could lead to new drugs against cancers driven by mutant RAS, and provided proof-of-principle for the phosphoestamer platform against PPIs in general.

cancer biology↗

bioRxiv2021

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

We previously discovered that deletion of c-Rel in the E-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-{kappa}B subunit in B-cell malignancies. Here we report that c-rel -/- E{micro}-Myc cells have an unexpected and major defect in the CHK1 pathway, with almost undetectable levels of CHK1 and CLSPN protein leading to therapeutic resistance to the highly specific CHK1 inhibitor (CHK1i) CCT244747. Similar downregulation of CHK1 levels was also seen in CCT244747 resistant U20S osteosarcoma cells. Further investigation revealed that downregulation of the deubiquitinase USP1 is responsible, at least in part, for these effects. Importantly, we demonstrate that c-rel -/- E{micro}-Myc lymphoma cells survive though upregulation of compensatory PI3K/AKT pathway activity. Moreover, targeting this pathway with Pictilisib (GDC-0941) effectively killed c-rel -/- E{micro}-Myc in vivo, while having no effect on wild type E-Myc cells. This data reveals an NF-{kappa}B regulated pathway controlling CHK1 activity in cancer cells and identifies a potential mechanism for both acquiring and overcoming CHK1i resistance in cancer patients.

cancer biology↗

bioRxiv2018

Regulation of checkpoint kinase signalling and tumorigenesis by the NF-κB regulated gene, CLSPN

Inhibition of the tumour promoting activities of NF-{kappa}B by cell signalling pathways has been proposed as a natural mechanism to limit the development of cancer. However, there has been a lack of evidence for these effects in vivo. Here we report that RelAT505A mice, where a CHK1 targeted Thr505 phosphosite is mutated to alanine, display earlier onset of MYC driven lymphoma than wild type littermates. We describe a positive feedback loop in which the NF-{kappa}B subunits RelA and c-Rel, in a manner dependent upon RelA Thr505 phosphorylation, drive the expression of the ATR checkpoint kinase regulator Claspin in response to DNA replication stress in cancer cells. This in turn is required for maintenance of CHK1 activity. Loss of a single allele of the Clspn gene in mice is sufficient to drive earlier tumorigenesis and low levels of CLSPN mRNA expression are associated with worse survival in some forms of human cancer. We propose that loss of this pathway early in tumorigenesis promotes cancer development through increased genomic instability. However, in malignant cancer cells it can help promote their addiction to the checkpoint kinase signalling required for the maintenance of genomic integrity. Importantly, disruption of this pathway leads to resistance of cells to treatment with CHK1 inhibitors. Claspin expression could therefore act as a biomarker for responsiveness of patients to CHK1 inhibitors and provide a potential pathway for the development of tumour resistance.

cancer biology↗