Biology subjects

Kane, J.

Publications and source records attributed to Kane, J..

5 recordsLinked to original sources

bioRxiv2023

Using a P. falciparum genetic cross to dissect the relative contributions of pfcrt and plasmepsin II/III to piperaquine response-related traits

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage isolate (KH004) and a drug susceptible parasite isolated in Malawi (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and four copies of pm2/3. We recovered 104 unique recombinant progeny and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3 for detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes, including PPQ survival assay (PSA), area under the dose-response curve (AUC), and a limited point IC50 (LP-IC50). We find that inheritance of the KH004 pfcrt allele is required for PPQ resistance, whereas copy number variation in pm2/3 further enhances resistance but does not confer resistance in the absence of PPQ-R-associated mutations in pfcrt. Deeper investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution in PPQ resistance. ImportanceResistance to PPQ used in combination with DHA has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention, and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and have the distinct advantage of assaying the effects of naturally evolved genetic variation. Our study was significantly strengthened because the full a range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multi-gene model suggests an important role for both loci in the evolution of this ACT resistant parasite lineage.

genomics↗

bioRxiv2023

A large-scale retrospective study in metastatic breast cancer patients using circulating tumor DNA and machine learning to predict treatment outcome and progression-free survival

PurposeMonitoring levels of circulating tumor-derived DNA (ctDNA) represents a non-invasive snapshot of tumor burden and potentially clonal evolution. Here we describe how a novel statistical model that uses serial ctDNA measurements from shallow whole genome sequencing (sWGS) in metastatic breast cancer patients produces a rapid and inexpensive assessment that is predictive of treatment response and progression-free survival. Patients and MethodsA cohort of 188 metastatic breast cancer patients had DNA extracted from serial plasma samples (total 1098, median=4, mean=5.87). Plasma DNA was assessed using sWGS and the tumor fraction in total cell free DNA estimated using ichorCNA. This approach was compared with ctDNA targeted sequencing and serial CA 15-3 measurements. The longitudinal ichorCNA values were used to develop a Bayesian learning model to predict subsequent treatment response. ResultsWe identified a transition point of 7% estimated tumor fraction to stratify patients into different categories of progression risk using ichorCNA estimates and a time-dependent Cox model, validated across different breast cancer subtypes and treatments, outperforming the alternative methods. We then developed a Bayesian learning model to predict subsequent treatment response with a sensitivity of 0.75 and a specificity of 0.66. ConclusionIn patients with metastatic breast cancer, sWGS of ctDNA and ichorCNA provide prognostic and predictive real-time valuable information on treatment response across subtypes and therapies. A prospective large-scale clinical trial to evaluate clinical benefit of early treatment changes based on ctDNA levels is now warranted.

genomics↗

bioRxiv2023

Modelling drug responses and evolutionary dynamics using triple negative breast cancer patient-derived xenografts

Triple negative breast cancers (TNBC) exhibit inter- and intra-tumour heterogeneity, which is reflected in diverse drug responses and interplays with tumour evolution. Here, we use TNBC patient-derived tumour xenografts (PDTX) as a platform for co-clinical trials to test their predictive value and explore the molecular features of drug response and resistance. Patients and their matched PDTX exhibited mirrored drug responses to neoadjuvant therapy in a clinical trial. In parallel, additional clinically-relevant treatments were tested in PDTXs in vivo to identify alternative effective therapies for each PDTX model. This framework establishes the foundation for anticipatory personalised therapies for those patients with resistant or relapsed tumours. The PDTXs were further explored to model PDTX- and treatment-specific behaviours. The dynamics of drug response were characterised at single-cell resolution revealing a novel mechanism of response to olaparib. Upon olaparib treatment PDTXs showed phenotypic plasticity, including transient activation of the immediate-early response and irreversible sequential phenotypic switches: from epithelial to epithelial-mesenchymal-hybrid states, and then to mesenchymal states. This molecular mechanism was exploited ex vivo by combining olaparib and salinomycin (an inhibitor of mesenchymal-transduced cells) to reveal synergistic effects. In summary, TNBC PDTXs have the potential to help design individualised treatment strategies derived from model-specific evolutionary insights.

cancer biology↗

bioRxiv2021

DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

Ewing sarcoma is the second most common bone malignancy in children and adolescents. Patients with upfront metastatic or recurrent disease have poor outcomes with 5-year survival rates of <30%. CDC7, also known as DDK (DBF4-dependent kinase), is a serine-threonine kinase that, in coordination with its activation subunit ASK (or DBF4), is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the replication stress response. Due to DDKs diverse roles during replication, coupled with an increased level of genomic instability and R-loop-mediated replication stress within Ewing sarcoma cells, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibitors. Here, we show that treatment with two selective DDK inhibitors, TAK-931 and XL413, results in apoptosis and a significant reduction in cell viability in EWS-FLI1-harboring Ewing sarcoma cell lines. We show that low dose DDK inhibition in Ewing sarcoma cells causes an accumulation of cells in late-S phase with a reduced replication capacity. There is also evidence of premature mitotic entry indicating an inability to properly complete DNA replication in a timely manner upon DDK inhibition. Also, there is a significant increase in the formation of micronuclei and other aberrant mitotic structures upon DDK inhibition in Ewing sarcoma cells indicating a failure to properly progress through S-phase followed by improper mitotic entry/progression, resulting in mitotic catastrophe. Interestingly, we observed minimal signs of mitotic accumulation, despite clear evidence of replication and mitotic stress, suggesting a failure to properly enforce the mitotic checkpoint. Together, these results suggest that Ewing sarcoma cells rely on the activity of DDK to maintain cell viability and suggest that DDK inhibition may prove to be a viable therapeutic strategy for patients with Ewing sarcoma.

cancer biology↗

bioRxiv2021

Transcriptional and translational responsiveness of the Neisseria gonorrhoeae type IV secretion system to conditions of host infections

The type IV secretion system of Neisseria gonorrhoeae translocates single-stranded DNA into the extracellular space, facilitating horizontal gene transfer and initiating biofilm formation. Expression of this system has been observed to be low under laboratory conditions, and multiple levels of regulation have been identified. We used a translational fusion of lacZ to traD, the gene for the type IV secretion system coupling protein, to screen for increased type IV secretion system expression. We identified several physiologically relevant conditions, including surface adherence, decreased manganese or iron, and increased zinc or copper, which increase gonococcal type IV secretion system protein levels through transcriptional and/or translational mechanisms. These metal treatments are reminiscent of the conditions in the macrophage phagosome. The ferric uptake regulator, Fur, was found to repress traD transcript levels, but to also have a second role, acting to allow TraD protein levels to increase only in the absence of iron. To better understand type IV secretion system regulation during infection, we examined transcriptomic data from active urethral infection samples from five men. These data demonstrated differential expression of 20 of 21 type IV secretion system genes during infection, indicating upregulation of genes necessary for DNA secretion during host infection.

microbiology↗