Target enzyme mutations are the molecular basis for resistance towards pharmacological inhibition of nicotinamide phosphoribosyltransferase

<p>Abstract</p> <p>Background</p> <p>Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) are promising cancer drugs currently in clinical trials in oncology, including APO866, CHS-828 and the CHS-828 prodrug EB1627/GMX1777, but cancer cell resistance to these drugs has not been studied in detail.</p> <p>Methods</p> <p>Here, we introduce an analogue of CHS-828 called TP201565 with increased potency in cellular assays. Further, we describe and characterize a panel of cell lines with acquired stable resistance towards several NAMPT inhibitors of 18 to 20,000 fold compared to their parental cell lines.</p> <p>Results</p> <p>We find that 4 out of 5 of the resistant sublines display mutations of NAMPT located in the vicinity of the active site or in the dimer interface of NAMPT. Furthermore, we show that these mutations are responsible for the resistance observed. All the resistant cell lines formed xenograft tumours <it>in vivo</it>. Also, we confirm CHS-828 and TP201565 as competitive inhibitors of NAMPT through docking studies and by NAMPT precipitation from cellular lysate by an analogue of TP201565 linked to sepharose. The NAMPT precipitation could be inhibited by addition of APO866.</p> <p>Conclusion</p> <p>We found that CHS-828 and TP201565 are competitive inhibitors of NAMPT and that acquired resistance towards NAMPT inhibitors can be expected primarily to be caused by mutations in NAMPT.</p

The genetic architecture of type 2 diabetes

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of heritability. To test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole genome sequencing in 2,657 Europeans with and without diabetes, and exome sequencing in a total of 12,940 subjects from five ancestral groups. To increase statistical power, we expanded sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support a major role for lower-frequency variants in predisposition to type 2 diabetes

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Publisher Correction: Sex-dimorphic genetic effects and novel loci for fasting glucose and insulin variability.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21276-3</jats:p

Methodological approaches in aggregate formation and microscopic analysis to assess pseudoislet morphology and cellular interactions

Microscopy has revolutionised our view on biology and has been vital for many discoveries since its invention around 200 years ago. Recent developments in cell biology have led to a strong interest in generating spheroids and organoids that better represent tissue. However, the current challenge faced by many researchers is the culture and analysis of these three-dimensional (3D) cell cultures. With the technological improvements in reconstructing volumetric datasets by optical sections, it is possible to quantify cells, their spatial arrangement, and the protein distribution without destroying the physical organization. We assessed three different microwell culture plates and four analysis tools for 3D imaging data for their applicability for the analysis of 3D cultures. A key advantage of microwell plates is their potential to perform high-throughput experiments in which cell cultures are generated and analysed in one single system. However, it was shown that this potential could be impacted by the material composition and microwell structure. For example, antibody staining was not possible in a hydrogel microwell, and truncated pyramid-structured microwells had increased background fluorescence due to their structure. Regarding analysis tools, four different software, namely CellProfiler, Fiji/ImageJ, Nikon GA3 and Imaris, were compared for their accuracy and applicability in analysing datasets from 3D cultures. The results showed that the open-access software, CellProfiler and Fiji, could quantify nuclei and cells, yet with varying results compared to manual counting, and may require post-processing optimisation. On the other hand, the GA3 and Imaris software packages showed excellent versatility in usage and accuracy in the quantification of nuclei and cells, and could classify cell localisation. Together these results provide critical considerations for microscopic imaging and analysis of 3D cell cultures