Biology subjects

Millar, V.

Publications and source records attributed to Millar, V..

3 recordsLinked to original sources

bioRxiv2022

Small-molecule Polθ inhibitors provide safe and effective tumor radiosensitization in preclinical models

DNA polymerase theta (Pol{theta}) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Pol{theta} has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Pol{theta} DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, normal fibroblasts are not radiosensitized by Pol{theta} inhibition. Mechanistically, we show that the radiosensitization caused by Pol{theta} inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Pol{theta} inhibitor with improved metabolic stability. In vivo, the combination of Pol{theta} inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared to radiation alone. These results pave the way for future clinical trials of Pol{theta} inhibitors in combination with radiotherapy.

cancer biology↗

bioRxiv2019

Germline and somatic genetic variants in the p53 pathway interact to affect cancer risk, progression and drug response

Insights into oncogenesis derived from cancer susceptibility loci could facilitate better cancer management and treatment through precision oncology. However, therapeutic applications have thus far been limited by our current lack of understanding regarding both their interactions with somatic cancer driver mutations and their influence on tumorigenesis. Here, by integrating germline datasets relating to cancer susceptibility with tumour data capturing somatically-acquired genetic variation, we provide evidence that single nucleotide polymorphism (SNPs) and somatic mutations in the p53 tumor suppressor pathway can interact to influence cancer development, progression and treatment response. We go on to provide human genetic evidence of a tumor-promoting role for the pro-survival activities of p53, which supports the development of more effective therapy combinations through their inhibition in cancers retaining wild-type p53. SignificanceWe describe significant interactions between heritable and somatic genetic variants in the p53 pathway that affect cancer susceptibility, progression and treatment response. Our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel therapeutic targets.

cancer biology↗

bioRxiv2017

Combined flow cytometry and high throughput image analysis for the study of essential genes in Caenorhabditis elegans

BackgroundThe advancement in automated image based microscopy platforms coupled with high throughput liquid workflows has facilitated the design of large scale screens utilizing multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high throughput approaches.\n\nResultsIn C. elegans, non-conditional lethal mutations can be maintained in heterozygosis using chromosome balancers, commonly labelled with GFP in the pharynx. Moreover gene-expression is typically monitored by the use of fluorescent reporters marked with the same fluorophore. Therefore, the separation of the different populations of animals at early larval stages represents a challenge. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at early larval stages. Because sorting is not completely error-free, we develop an automated high-throughput image-analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image-analysis in a functional genomic RNAi screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while, homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both, known and new PHB genetic interactors affecting the UPRmt and growth.\n\nConclusionsA systematic way to analyse genetic interactions of essential genes in multicellular organisms is lacking. The method presented here allows the study of balanced lethal mutations in a high-throughput manner and can be easily adapted depending on the users requirements. Therefore, it will serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

genetics↗