Phosphoinositide 3‐kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signaling, leading to cell survival ...and proliferation. Phosphorylation of Akt Thr308 by PI3K‐PDK1 and Akt Ser473 by mammalian target of rapamycin complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K‐activated Akt signaling is well established in HNSCC, the significance of mTORC2‐driven Akt signaling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2‐specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy.
While the role of phosphoinositide 3‐kinase (PI3K)‐activated Akt signalling is well established in head/neck squamous cell carcinogenesis, the significance of mammalian target of rapamycin complex 2 (mTORC2)‐driven Akt signalling has not been thoroughly examined. Our study reveals an oncogenic role for mTORC2 and its obligate subunit RICTOR. Disruption of RICTOR/mTORC2 signalling impairs cellular viability and enhances the efficacy of PI3K targeted inhibition. Collectively, these findings rationalize the development of an mTORC2‐specific inhibitor.
Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular ...diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1 / BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5′ and 3′ of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective ( n = 95) and prospective ( n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants.
Human apolipoprotein (apo) C‐II is one of several plasma apolipoproteins that form amyloid deposits in vivo and is an independent risk factor for cardiovascular disease. Lipid‐free apoC‐II readily ...self‐assembles into twisted‐ribbon amyloid fibrils but forms straight, rod‐like amyloid fibrils in the presence of low concentrations of micellar phospholipids. Charge mutations exerted significantly different effects on rod‐like fibril formation compared to their effects on twisted‐ribbon fibril formation. For instance, the double mutant, K30D‐D69K apoC‐II, readily formed twisted‐ribbon fibrils, while the rate of rod‐like fibril formation in the presence of micellar phospholipid was negligible. Structural analysis of rod‐like apoC‐II fibrils, using hydrogen–deuterium exchange and NMR analysis showed exchange protection consistent with a core cross‐β structure comprising the C‐terminal 58–76 region. Molecular dynamics simulations of fibril arrangements for this region favoured a parallel cross‐β structure. X‐ray fibre diffraction data for aligned rod‐like fibrils showed a major meridional spacing at 4.6 Å and equatorial spacings at 9.7, 23.8 and 46.6 Å. The latter two equatorial spacings are not observed for aligned twisted‐ribbon fibrils and are predicted for a model involving two cross‐β fibrils in an off‐set antiparallel structure with four apoC‐II units per rise of the β‐sheet. This model is consistent with the mutational effects on rod‐like apoC‐II fibril formation. The lipid‐dependent polymorphisms exhibited by apoC‐II fibrils could determine the properties of apoC‐II in renal amyloid deposits and their potential role in the development of cardiovascular disease.
Human apolipoprotein (apo) C‐II can form two distinct types of amyloid fibrils, depending on the presence and nature of lipid in solution. We have investigated the structure of ‘rod‐like’ apoC‐II amyloid fibrils using biophysical and computational methods, and propose a model for the core cross‐β structure of the fibrils.
Diabetes affects select organs such as the eyes, kidney, heart, and brain. Our recent studies show that diabetes also enhances adipogenesis in the bone marrow and reduces the number of ...marrow-resident vascular regenerative stem cells. In the current study, we have performed a detailed spatio-temporal examination to identify the early changes that are induced by diabetes in the bone marrow. Here we show that short-term diabetes causes structural and molecular changes in the marrow, including enhanced adipogenesis in tibiae of mice, prior to stem cell depletion. This enhanced adipogenesis was associated with suppressed transforming growth factor-beta (TGFB) signaling. Using human bone marrow-derived mesenchymal progenitor cells, we show that TGFB pathway suppresses adipogenic differentiation through TGFB-activated kinase 1 (TAK1). These findings may inform the development of novel therapeutic targets for patients with diabetes to restore regenerative stem cell function.
The sinus node inhibitor ivabradine was approved for patients with heart failure (HF) after the ivabradine and outcomes in chronic HF (SHIFT Systolic Heart Failure Treatment With the IF Inhibitor ...Ivabradine Trial) trial. Our objective was to characterize the proportion of patients with HF eligible for ivabradine and the representativeness of the SHIFT trial enrollees compared with those in the Swedish Heart Failure Registry.
We examined 26 404 patients with clinical HF from the Swedish Heart Failure Registry and divided them into SHIFT type (left ventricular ejection fraction <40%, New York Heart Association class II-IV, sinus rhythm, and heart rate ≥70 beats per minute) and non-SHIFT type. Baseline characteristics and medication use were compared and change in eligibility over time was reported at 6 months and 1 year in a subset of patients. Overall, 14.2% (n=3741) of patients were SHIFT type. These patients were more likely to be younger, men, have diabetes mellitus, ischemic heart disease, lower left ventricular ejection fraction, and more recent onset HF (<6 months; all,
<0.001). Although 88.9% of SHIFT type and 88.5% of non-SHIFT type (
=0.421) were receiving selected β-blockers, only 58.8% and 67.3% (
<0.001) were on >50% of target dose. From those patients who had repeated visits within 6 months (n=5420) and 1 year (n=6840), respectively, 10.2% (n=555) and 10.6% (n=724) of SHIFT-type patients became ineligible, 77.3% (n=4188) and 77.3% (n=5287) remained ineligible, and 4.6% (n=252) and 4.9% (n=335) of non-SHIFT-type patients became eligible for initiation of ivabradine.
From the Swedish Heart Failure Registry, 14.2% of patients with HF were eligible for ivabradine. These patients more commonly were not receiving target β-blocker dose. Over time, a minority of patients became ineligible and an even smaller minority became eligible.
Paroxysmal atrial fibrillation (PAF) is the most common cardiac arrhythmia, conveying a stroke risk comparable to persistent AF. It poses a significant diagnostic challenge given its intermittency ...and potential brevity, and absence of symptoms in most patients. This pilot study introduces a novel biomarker for early PAF detection, based upon analysis of sinus rhythm ECG waveform complexity. Sinus rhythm ECG recordings were made from 52 patients with (
= 28) or without (
= 24) a subsequent diagnosis of PAF. Subjects used a handheld ECG monitor to record 28-second periods, twice-daily for at least 3 weeks. Two independent ECG complexity indices were calculated using a Lempel-Ziv algorithm: R-wave interval variability (beat detection, BD) and complexity of the entire ECG waveform (threshold crossing, TC). TC, but not BD, complexity scores were significantly greater in PAF patients, but TC complexity alone did not identify satisfactorily individual PAF cases. However, a composite complexity score (
-score) based on within-patient BD and TC variability scores was devised. The
-score allowed correct identification of PAF patients with 85% sensitivity and 83% specificity. This powerful but simple approach to identify PAF sufferers from analysis of brief periods of sinus-rhythm ECGs using hand-held monitors should enable easy and low-cost screening for PAF with the potential to reduce stroke occurrence.
Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Despite pre-clinical and clinical ...studies, outcomes from targeting the PI3K pathway have been underwhelming and the development of drug resistance poses a significant barrier to patient treatment. In the present study, we examined mechanisms of acquired resistance to the PI3Kα inhibitor alpelisib (formerly BYL719) in HNSCC cell lines and patient-derived xenografts (PDXs).
Five unique PDX mouse models and three HNSCC cell lines were used. All cell lines and xenografts underwent genomic characterization prior to study. Serial drug treatment was conducted in vitro and in vivo to develop multiple, clinically-significant models of resistance to alpelisib. We then used reverse phase protein arrays (RPPAs) to profile the expression of proteins in parental and drug-resistant models. Top hits were validated by immunoblotting and immunohistochemistry. Flow cytometric analysis and RNA interference studies were then used to interrogate the molecular mechanisms underlying acquired drug resistance.
Prolonged treatment with alpelisib led to upregulation of TAM family receptor tyrosine kinases TYRO3 and AXL. Importantly, a significant shift in expression of both TYRO3 and AXL to the cell surface was detected in drug-resistant cells. Targeted knockdown of TYRO3 and AXL effectively re-sensitized resistant cells to PI3Kα inhibition. In vivo, resistance to alpelisib emerged following 20-35 days of treatment in all five PDX models. Elevated TYRO3 expression was detected in drug-resistant PDX tissues. Downstream of TYRO3 and AXL, we identified activation of intracellular MAPK signalling. Inhibition of MAPK signalling also re-sensitized drug-resistant cells to alpelisib.
We have identified TYRO3 and AXL receptors to be key mediators of resistance to alpelisib, both in vitro and in vivo. Our findings suggest that pan-TAM inhibition is a promising avenue for combinatorial or second-line therapy alongside PI3Kα inhibition. These findings advance our understanding of the role TAM receptors play in modulating the response of HNSCC to PI3Kα inhibition and suggest a means to prevent, or at least delay, resistance to PI3Kα inhibition in order to improve outcomes for HNSCC patients.
Aim
To develop a pyrosequencing assay to monitor the frequency of alleles of an avirulence gene, AvrLm4, in populations of sexual spores of Leptosphaeria maculans, a fungal pathogen of canola ...(Brassica napus).
Methods and Results
The predominant mutation in AvrLm4 responsible for virulence to the corresponding resistance gene, Rlm4, is a single nucleotide polymorphism (SNP) at base 358. Pyrosequencing primers were designed to amplify a 90‐bp region that included this SNP. The assay was developed and validated by analysing the frequency of AvrLm4 in isolate mixtures of different proportions. Furthermore, the frequency of avrLm4 (virulence allele) determined by pyrosequencing of populations of sexual spores was consistent with the frequency of avrLm4 determined by Sanger sequencing of the entire AvrLm4 gene from single isolates cultured from the same stubble.
Conclusion
This high‐throughput assay can play an important role in predicting the risk of resistance breakdown in crops.
Significance and Impact of the Study
Similar assays can be applied to monitor frequencies of fungicide resistance in pathogens of crops and to assay diversity in microbial soil communities such as in soil samples from bat caves where white‐nose syndrome has been detected.
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