Biology subjects

Grimsley, H. E.

Publications and source records attributed to Grimsley, H. E..

5 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

The 9-1-1 complex protects ssDNA gaps in BRCA2-deficient cancer

Single-stranded DNA (ssDNA) gaps are a hallmark of BRCA-deficient cells, yet the mechanisms that safeguard these lesions remain unclear. Through a genome-wide CRISPR screen, we identified the RAD9A-HUS1-RAD1 (9-1-1) complex as essential for the survival of BRCA2-deficient cells through an ATR-independent mechanism. Loss of 9-1-1 in this context leads to the accumulation of PRIMPOL-dependent gaps that fail to undergo post-replicat ive repair, resulting in pathological expansion and increased DNA damage. This instability is driven by excessive EXO1-mediated degradation, as EXO1 depletion rescues the phenotype. We further demonstrate that the 9-1-1 complex is required for POL{zeta}-dependent gap filling. We propose a model in which ssDNA gaps, when extended beyond a critical length, become inaccessible to TLS-mediated repair and are fully reliant on homologous recombination. These findings establish the 9-1-1 complex as key regulator of ssDNA gap stability and a promising therapeutic target in BRCA2-deficient cancers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/680950v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@56516aorg.highwire.dtl.DTLVardef@fb1ce3org.highwire.dtl.DTLVardef@25a7b2org.highwire.dtl.DTLVardef@249731_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Resistance patterns in drug-adapted cancer cell lines reflect complex evolution in clinical tumors

BackgroundHere, we introduce a novel set of triple-negative breast cancer (TNBC) cell lines consisting of MDA-MB-468, HCC38, and HCC1806 and their sublines adapted to cisplatin, doxorubicin, eribulin, paclitaxel, gemcitabine, or 5-fluorouracil. MethodsThe cell lines were characterized by whole exome sequencing and the determination of drug-response profiles. Moreover, genes harbouring resistance-associated mutations were investigated using TCGA data for potential clinical relevance. ResultSequencing combined with TCGA-derived patient data resulted in the identification of 682 biomarker candidates in the pan-cancer analysis. Thirty-five genes were considered the most promising candidates because they harboured resistance-associated variants in at least two resistant sublines, and their expression correlated with TNBC patient survival. Exome sequencing and response profiles to cytotoxic drugs and DNA damage response inhibitors identified revealed remarkably little overlap between the resistant sublines, suggesting that each resistance formation process follows a unique route. All of the drug-resistant TNBC sublines remained sensitive or even displayed collateral sensitivity to a range of tested compounds. Cross-resistance levels were lowest for the CHK2 inhibitor CCT241533, the PLK1 inhibitor SBE13, and the RAD51 recombinase inhibitor B02, suggesting that CHK2, PLK1, and RAD51 are potential drug targets for therapy-refractory TNBC. ConclusionsWe present novel preclinical models of acquired drug resistance in TNBC and many novel candidate biomarkers for further investigation. The finding that each cancer cell line adaptation process follows an unpredictable route reflects recent findings on cancer cell evolution in patients, supporting the relevance of drug-adapted cancer cell lines as preclinical models of acquired resistance.

cancer biology↗

bioRxiv2023

A local ATR-dependent checkpoint pathway is activated by a site-specific replication fork block in human cells

When replication forks encounter DNA lesions that cause polymerase stalling a checkpoint pathway is activated. The ATR-dependent intra-S checkpoint pathway mediates detection and processing of sites of replication fork stalling to maintain genomic integrity. Several factors involved in the global checkpoint pathway have been identified, but the response to a single replication fork barrier (RFB) is poorly understood. We utilized the E.coli-based Tus-Ter system in human MCF7 cells and showed that the Tus protein binding to TerB sequences creates an efficient site-specific RFB. The single fork RFB was sufficient to activate a local, but not global, ATR-dependent checkpoint response that leads to phosphorylation and accumulation of DNA damage sensor protein {gamma}H2AX, confined locally to within a kilobase of the site of stalling. These data support a model of local management of fork stalling, which allows global replication at sites other than the RFB to continue to progress without delay.

molecular biology↗

bioRxiv2019

A novel exosome biogenesis mechanism: multivesicular structures budding and rupturing at the plasma membrane

Exosomes are small vesicles secreted by the cells, which mediate intercellular signalling and systemic physiological processes. Exosomes are known to originate from the intraluminal vesicles of the multivesicular endosome that fuses with the plasma membrane. We found that the non-small cell lung cancer (NSCLC) cell lines, HCC15 and A549, secreted exosomes with typical morphology and protein contents. Unexpectedly, transmission electron microscopy images indicated that the cells formed multivesicular structures that protruded from the plasma membrane and ruptured to release the exosomes. There were smooth multivesicular structures surrounded by an ordinary looking membrane, multivesicular structures coated by an electron dense layer with regular spacing pattern, and intermediate forms that combined elements of both. Electron microscopy images suggested that exosomes are release from these structures by burst events and not by the conventional fusion process. The molecular details of this novel mechanism for membrane association, deformation and fusion is to be unveiled in the future.

cell biology↗