Mutation of threonine 766 in the epidermal growth factor receptor reveals a hotspot for resistance formation against selective tyrosine kinase inhibitors

Small molecule inhibitors of protein tyrosine kinases such as STI571 represent a major new class of therapeutics for target-selective treatment of human cancer. Clinical resistance formation to the BCR-ABL inhibitor STI571 has been observed in patients with advanced chronic myeloid leukemia and was frequently caused by a C to T single nucleotide change in the Abl kinase domain, which substituted Thr-315 with isoleucine and rendered BCR-ABL resistant to STI571 inhibition. The corresponding mutation in the epidermal growth factor receptor (EGFR) tyrosine kinase replaced Thr-766 of the EGFR by methionine and dramatically reduced the sensitivity of EGFR to inhibition by selective 4-anilinoquinazoline inhibitors such as PD153035. Inhibitor-resistant EGFR exhibited the same signaling capacity as wild-type receptor in vivo and provides a useful tool for analyzing EGFR-mediated signal transduction. Our data identify Thr-766 of the EGFR as a structural determinant that bears the potential to become a relevant feature in resistance formation during cancer therapy with EGFR-specific 4-anilinoquinazoline inhibitors.     

Activated epidermal growth factor receptor as a novel target in pancreatic cancer therapy

Pancreatic cancer is one of the most fatal among all solid malignancies. Targeted therapeutic approaches have the potential to transform cancer therapy as exemplified by the success of several tyrosine kinase inhibitors. Prompted by this, comprehensive profiling of tyrosine kinases and their substrates was carried out using a panel of low passage pancreatic cancer cell lines. One of the pancreatic cancer cell lines, P196, which showed dramatic upregulation of tyrosine kinase activity as compared to non-neoplastic cells, was systematically studied using a quantitative proteomic approach called stable isotope labeling with amino acids in cell culture (SILAC). A careful analysis of activated tyrosine kinase pathways revealed aberrant activation of epidermal growth factor receptor pathway in this cell line. Mouse xenograft based studies using EGFR inhibitor erlotinib confirmed EGFR pathway to be responsible for proliferation in these tumors. By a systematic study across low passage pancreatic cancer cell lines and mice carrying pancreatic cancer xenografts, we have demonstrated activated epidermal growth factor receptor as an attractive candidate for targeted therapy in a subset of pancreatic cancers. Further, we propose immunohistochemical labeling of activated EGFR (pEGFR (1068)) as an efficient screening tool to select patients who are more likely to respond to EGFR inhibitors.     

NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m⁵C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.     

Circadiane Uhren und Energiemetabolismus

Chrononutrition – circadian clocks and energy metabolism Genetically encoded endogenous clocks regulate 24‐hour rhythms of physiology and behavior. A central pacemaker residing in the suprachiasmatic nucleus synchronizes peripheral clocks found in all tissues with each other and with the external day‐night cycle. One function of circadian clocks is the regulation of energy metabolism via rhythmic activation of tissue‐specific clock‐controlled genes. In the liver, genes involved in glucose and lipid metabolism are regulated in this fashion, while in adipocytes, fatty acid release and adipokine secretion are controlled by the circadian clock. Disruption of circadian rhythms as seen, for example, in shift workers promotes the development of metabolic disorders such as obesity and type‐2 diabetes.     

SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation and gene repression

Polycomb group (PcG) complexes regulate cellular identity through epigenetic programming of chromatin. Here, we show that SSX2, a germline-specific protein ectopically expressed in melanoma and other types of human cancers, is a chromatin-associated protein that antagonizes BMI1 and EZH2 PcG body formation and derepresses PcG target genes. SSX2 further negatively regulates the level of the PcG-associated histone mark H3K27me3 in melanoma cells, and there is a clear inverse correlation between SSX2/3 expression and H3K27me3 in spermatogenesis. However, SSX2 does not affect the overall composition and stability of PcG complexes, and there is no direct concordance between SSX2 and BMI1/H3K27me3 presence at regulated genes. This suggests that SSX2 antagonizes PcG function through an indirect mechanism, such as modulation of chromatin structure. SSX2 binds double-stranded DNA in a sequence non-specific manner in agreement with the observed widespread association with chromatin. Our results implicate SSX2 in regulation of chromatin structure and function.     

Soluble Urokinase Plasminogen Activator Receptor as a Marker for Use of Antidepressants

Objectives

Inflammation is involved in the pathogenesis of depression. A few cross-sectional population-based studies have found that depression is associated with increased levels of inflammatory markers. Soluble urokinase plasminogen activation receptor (suPAR) is known to be a stable marker for inflammation. We investigated the bidirectional association between suPAR levels and use of antidepressants.

Methods

suPAR level was measured in 9305 blood donors and analysed in relation to 5-years follow-up data on purchase of antidepressants and hospital diagnoses of depression from a nationwide Danish register.

Results

For men and women without prior use of antidepressants we found a significantly higher risk for incident use of antidepressants with higher suPAR values. For men, the risk of first use of antidepressants increased by 72% from the 1^st to the 4^th quartile (HR = 1.72, 95% CI: 1.11–2.69). For women, it increased by 108% from the 1^st to the 4^th quartile (HR = 2.08, 95% CI: 1.45–2.98). Previous use of antidepressants was also significantly associated with higher suPAR levels (p = 0.002).

Conclusions

High suPAR levels are associated with an increased risk for both previous and future use of antidepressants in healthy men and women. High suPAR are also associated with increased risk for a hospital diagnosis of depression.     

Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module.

The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.     

Pigmentary Markers in Danes – Associations with Quantitative Skin Colour,Nevi Count,Familial Atypical Multiple-Mole,and Melanoma Syndrome

To investigate whether pigmentation genes involved in the melanogenic pathway (melanogenesis) contributed to melanoma predisposition, we compared pigmentary genetics with quantitative skin pigmentation measurements, the number of atypical nevi, the total nevus count, and the familial atypical multiple mole and melanoma (FAMMM) syndrome. We typed 32 pigmentary SNP markers and sequenced MC1R in 246 healthy individuals and 116 individuals attending periodic control for malignant melanoma development, 50 of which were diagnosed with FAMMM. It was observed that individuals with any two grouped MC1R variants (missense, {"type":"entrez-nucleotide","attrs":{"text":"NM_002386","term_id":"193083133"}}NM_002386:c. 456C > A (p.TYR152*), or {"type":"entrez-nucleotide","attrs":{"text":"NM_002386","term_id":"193083133"}}NM_002386:c.83_84insA (p.Asn29Glnfs*14) had significantly (p<0.001) lighter skin pigmentation of the upper-inner arm than those with none or one MC1R variant. We did not observe any significant association of the MC1R variants with constitutive pigmentation measured on the buttock area. We hypothesize that the effect of MC1R variants on arm pigmentation is primarily reflecting the inability to tan when subjected to UVR. A gender specific effect on skin pigmentation was also observed, and it was found that the skin pigmentation of females on average were darker than that of males (p<0.01). We conclude that MC1R variants are associated with quantitative skin colour in a lightly pigmented Danish population. We did not observe any association between any pigmentary marker and the FAMMM syndrome. We suggest that the genetics of FAMMM is not related to the genetics of the pigmentary pathway.     

Culturable prokaryotic diversity of deep,gas hydrate sediments: first use of a continuous high‐pressure,anaerobic, enrichment and isolation system for subseafloor sediments (DeepIsoBUG)

Deep subseafloor sediments may contain depressurization‐sensitive, anaerobic, piezophilic prokaryotes. To test this we developed the DeepIsoBUG system, which when coupled with the HYACINTH pressure‐retaining drilling and core storage system and the PRESS core cutting and processing system, enables deep sediments to be handled without depressurization (up to 25 MPa) and anaerobic prokaryotic enrichments and isolation to be conducted up to 100 MPa. Here, we describe the system and its first use with subsurface gas hydrate sediments from the Indian Continental Shelf, Cascadia Margin and Gulf of Mexico. Generally, highest cell concentrations in enrichments occurred close to in situ pressures (14 MPa) in a variety of media, although growth continued up to at least 80 MPa. Predominant sequences in enrichments were Carnobacterium, Clostridium, Marinilactibacillus and Pseudomonas, plus Acetobacterium and Bacteroidetes in Indian samples, largely independent of media and pressures. Related 16S rRNA gene sequences for all of these Bacteria have been detected in deep, subsurface environments, although isolated strains were piezotolerant, being able to grow at atmospheric pressure. Only the Clostridium and Acetobacterium were obligate anaerobes. No Archaea were enriched. It may be that these sediment samples were not deep enough (total depth 1126–1527 m) to obtain obligate piezophiles.