BACKGROUND: Emerging evidence suggests that tumour morphological heterogeneity may influence mutational profiles relevant to therapy response. In this pilot study, we aimed to assess whether mutations identified within specific morphological patterns or at the invasion front correlate with shorter time to progression after anti-EGFR therapy, as compared to whole-tissue analysis. METHODS: We investigated genetic mutations in 142 samples from primary tumours of 39 KRAS wild-type metastatic colorectal cancer (CRC) patients receiving anti-EGFR therapy. Deep next-generation sequencing was performed on whole-tumour sections and six morphology-defined tumour regions. RESULTS: Mutations in genes linked to anti-EGFR therapy response (KRAS, BRAF, NRAS, PTEN and PI3KCA) were found uniquely in the non-responder group, with substantial variability across morphological sub-regions. BRAF mutations were aligned with serrated and mucinous morphologies, while KRAS mutations (p.Lys147Glu and p.Ala146Thr) were associated with mucinous and desmoplastic morphologies. In all cases, the cumulative mutational profile from sub-regions provided more details than that of the whole-tumour profile. CONCLUSION: Our findings highlight that comprehensive analysis, considering morphological heterogeneity, is crucial for personalised CRC treatment strategies.

• Klíčová slova
• BRAF, KRAS, NRAS, PIK3CA, PTEN, anti‐EGFR therapy, invasion front, metastatic cancer, morphological sampling, tumour heterogeneity,
• MeSH
• chemorezistence * genetika   MeSH
• dospělí MeSH
• erbB receptory antagonisté a inhibitory   MeSH
• fosfohydroláza PTEN genetika   MeSH
• GTP-fosfohydrolasy genetika   MeSH
• inhibitory proteinkinas * terapeutické užití   MeSH
• kolorektální nádory * genetika   farmakoterapie   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• pilotní projekty MeSH
• protinádorové látky * terapeutické užití   MeSH
• protoonkogenní proteiny B-Raf genetika   MeSH
• protoonkogenní proteiny p21(ras) genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• BRAF protein, human MeSH Prohlížeč
• EGFR protein, human MeSH Prohlížeč
• erbB receptory MeSH
• fosfohydroláza PTEN MeSH
• GTP-fosfohydrolasy MeSH
• inhibitory proteinkinas * MeSH
• KRAS protein, human MeSH Prohlížeč
• protinádorové látky * MeSH
• protoonkogenní proteiny B-Raf MeSH
• protoonkogenní proteiny p21(ras) MeSH

Specific patterns of mitochondrial dynamics have been repeatedly reported to promote drug resistance in cancer. However, whether targeting mitochondrial fission- and fusion-related proteins could be leveraged to combat multidrug-resistant pediatric sarcomas is poorly understood. Here, we demonstrated that the expression and activation of the mitochondrial fission mediator DRP1 are affected by chemotherapy exposure in common pediatric sarcomas, namely, rhabdomyosarcoma and osteosarcoma. Unexpectedly, decreasing DRP1 activity through stable DRP1 knockdown neither attenuated sarcoma drug resistance nor affected growth rate or mitochondrial network morphology. The minimal impact on sarcoma cell physiology, along with the up-regulation of fission adaptor proteins (MFF and FIS1) detected in rhabdomyosarcoma cells, suggests an alternative DRP1-independent mitochondrial fission mechanism that may efficiently compensate for the lack of DRP1 activity. By exploring the upstream mitophagy and mitochondrial fission regulator, AMPKα1, we found that markedly reduced AMPKα1 levels are sufficient to maintain AMPK signaling capacity without affecting chemosensitivity. Collectively, our findings challenge the direct involvement of DRP1 in pediatric sarcoma drug resistance and highlight the complexity of yet-to-be-characterized noncanonical regulators of mitochondrial dynamics.

• MeSH
• chemorezistence * genetika   MeSH
• dynaminy * metabolismus   genetika   MeSH
• GTP-fosfohydrolasy metabolismus   genetika   MeSH
• lidé MeSH
• membránové proteiny metabolismus   genetika   MeSH
• mitochondriální dynamika * účinky léků   MeSH
• mitochondriální proteiny * metabolismus   genetika   MeSH
• mitochondrie * metabolismus   účinky léků   MeSH
• mitofagie účinky léků   genetika   MeSH
• nádorové buněčné linie MeSH
• osteosarkom metabolismus   patologie   farmakoterapie   genetika   MeSH
• proteiny asociované s mikrotubuly metabolismus   genetika   MeSH
• protinádorové látky farmakologie   MeSH
• rhabdomyosarkom metabolismus   genetika   patologie   MeSH
• sarkom * metabolismus   genetika   farmakoterapie   patologie   MeSH
• signální transdukce účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNM1L protein, human MeSH Prohlížeč
• dynaminy * MeSH
• FIS1 protein, human MeSH Prohlížeč
• GTP-fosfohydrolasy MeSH
• membránové proteiny MeSH
• Mff protein, human MeSH Prohlížeč
• mitochondriální proteiny * MeSH
• proteiny asociované s mikrotubuly MeSH
• protinádorové látky MeSH

The evolution of eukaryotes is a fundamental event in the history of life. The closest prokaryotic lineage to eukaryotes, the Asgardarchaeota, encode proteins previously found only in eukaryotes, providing insight into their archaeal ancestor. Eukaryotic cells are characterized by endomembrane organelles, and the Arf family GTPases regulate organelle dynamics by recruiting effector proteins to membranes upon activation. The Arf family is ubiquitous among eukaryotes, but its origins remain elusive. Here we report a group of prokaryotic GTPases, the ArfRs, which are widely present in Asgardarchaeota. Phylogenetic analyses reveal that eukaryotic Arf family proteins arose from the ArfR group. Expression of representative Asgardarchaeota ArfR proteins in yeast and X-ray crystallographic studies show that ArfR GTPases possess the mechanism of membrane binding and structural features unique to Arf family proteins. Our results indicate that Arf family GTPases originated in the archaeal ancestor of eukaryotes, consistent with aspects of the endomembrane system evolving early in eukaryogenesis.

• MeSH
• Archaea * genetika   enzymologie   klasifikace   MeSH
• archeální proteiny * genetika   metabolismus   chemie   MeSH
• Eukaryota * genetika   enzymologie   MeSH
• eukaryotické buňky * MeSH
• fylogeneze MeSH
• GTP-fosfohydrolasy * genetika   metabolismus   chemie   MeSH
• krystalografie rentgenová MeSH
• molekulární evoluce * MeSH
• organely * metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• archeální proteiny * MeSH
• GTP-fosfohydrolasy * MeSH

The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations.

• Klíčová slova
• DUSP6, ERK, MAP kinase, eIF4F, melanoma,
• MeSH
• eukaryotický iniciační faktor 4F * metabolismus   genetika   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosfatasa 6 s dvojí specificitou metabolismus   genetika   MeSH
• GTP-fosfohydrolasy * metabolismus   genetika   MeSH
• lidé MeSH
• MAP kinasový signální systém * genetika   MeSH
• melanom * genetika   metabolismus   patologie   MeSH
• membránové proteiny * metabolismus   genetika   MeSH
• mutace * MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• protoonkogenní proteiny B-Raf * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• BRAF protein, human MeSH Prohlížeč
• DUSP6 protein, human MeSH Prohlížeč
• EIF4E protein, human MeSH Prohlížeč
• eukaryotický iniciační faktor 4F * MeSH
• extracelulárním signálem regulované MAP kinasy MeSH
• fosfatasa 6 s dvojí specificitou MeSH
• GTP-fosfohydrolasy * MeSH
• membránové proteiny * MeSH
• NRAS protein, human MeSH Prohlížeč
• protoonkogenní proteiny B-Raf * MeSH

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

• Klíčová slova
• NF1, RAS Interactome, RAS Signaling, Senescence-Associated Secretory Phenotype, Ubiquitination,
• MeSH
• GTP-fosfohydrolasy metabolismus   genetika   MeSH
• lidé MeSH
• lysin metabolismus   MeSH
• membránové proteiny metabolismus   genetika   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• neurofibromin 1 MeSH
• protein aktivující GTPasu p120 metabolismus   genetika   MeSH
• protein-serin-threoninkinasy metabolismus   genetika   MeSH
• protoonkogenní proteiny p21(ras) * metabolismus   genetika   MeSH
• Ras proteiny metabolismus   genetika   MeSH
• signální transdukce MeSH
• ubikvitinace * MeSH
• vazba proteinů * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• GTP-fosfohydrolasy MeSH
• KRAS protein, human MeSH Prohlížeč
• lysin MeSH
• membránové proteiny MeSH
• neurofibromin 1 MeSH
• NF1 protein, human MeSH Prohlížeč
• NRAS protein, human MeSH Prohlížeč
• protein aktivující GTPasu p120 MeSH
• protein-serin-threoninkinasy MeSH
• protoonkogenní proteiny p21(ras) * MeSH
• Ras proteiny MeSH
• RASA1 protein, human MeSH Prohlížeč
• TBK1 protein, human MeSH Prohlížeč

Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse.

• MeSH
• anaplastická lymfomová kináza * genetika   metabolismus   antagonisté a inhibitory   MeSH
• apoptóza účinky léků   genetika   MeSH
• chemorezistence genetika   účinky léků   MeSH
• dibenzocyklohepteny * MeSH
• farnesyltranstransferasa * antagonisté a inhibitory   metabolismus   MeSH
• GTP-fosfohydrolasy * genetika   metabolismus   MeSH
• inhibitory proteinkinas * farmakologie   terapeutické užití   MeSH
• lidé MeSH
• membránové proteiny metabolismus   genetika   MeSH
• mikro RNA * genetika   metabolismus   MeSH
• mutace MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• neuroblastom * farmakoterapie   genetika   patologie   metabolismus   MeSH
• piperidiny * farmakologie   terapeutické užití   MeSH
• pyridiny * farmakologie   terapeutické užití   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• synergismus léků MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ALK protein, human MeSH Prohlížeč
• anaplastická lymfomová kináza * MeSH
• dibenzocyklohepteny * MeSH
• farnesyltranstransferasa * MeSH
• GTP-fosfohydrolasy * MeSH
• inhibitory proteinkinas * MeSH
• lonafarnib MeSH Prohlížeč
• membránové proteiny MeSH
• mikro RNA * MeSH
• NRAS protein, human MeSH Prohlížeč
• piperidiny * MeSH
• pyridiny * MeSH

• MeSH
• akutní myeloidní leukemie * diagnóza   genetika   MeSH
• bodová mutace MeSH
• GTP-fosfohydrolasy genetika   MeSH
• lidé MeSH
• membránové proteiny genetika   MeSH
• mutace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GTP-fosfohydrolasy MeSH
• membránové proteiny MeSH
• NRAS protein, human MeSH Prohlížeč

The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas® KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA.

• MeSH
• exony MeSH
• GTP-fosfohydrolasy genetika   MeSH
• kolorektální nádory * genetika   MeSH
• lidé MeSH
• membránové proteiny genetika   MeSH
• mutace MeSH
• protoonkogenní proteiny p21(ras) * genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• GTP-fosfohydrolasy MeSH
• KRAS protein, human MeSH Prohlížeč
• membránové proteiny MeSH
• NRAS protein, human MeSH Prohlížeč
• protoonkogenní proteiny p21(ras) * MeSH

The evolution of eukaryotic cellular complexity is interwoven with the extensive diversification of many protein families. One key family is the ARF GTPases that act in eukaryote-specific processes, including membrane traffic, tubulin assembly, actin dynamics, and cilia-related functions. Unfortunately, our understanding of the evolution of this family is limited. Sampling an extensive set of available genome and transcriptome sequences, we have assembled a data set of over 2,000 manually curated ARF family genes from 114 eukaryotic species, including many deeply diverged protist lineages, and carried out comprehensive molecular phylogenetic analyses. These reconstructed as many as 16 ARF family members present in the last eukaryotic common ancestor, nearly doubling the previously inferred ancient system complexity. Evidence for the wide occurrence and ancestral origin of Arf6, Arl13, and Arl16 is presented for the first time. Moreover, Arl17, Arl18, and SarB, newly described here, are absent from well-studied model organisms and as a result their function(s) remain unknown. Analyses of our data set revealed a previously unsuspected diversity of membrane association modes and domain architectures within the ARF family. We detail the step-wise expansion of the ARF family in the metazoan lineage, including discovery of several new animal-specific family members. Delving back to its earliest evolution in eukaryotes, the resolved relationship observed between the ARF family paralogs sets boundaries for scenarios of vesicle coat origins during eukaryogenesis. Altogether, our work fundamentally broadens the understanding of the diversity and evolution of a protein family underpinning the structural and functional complexity of the eukaryote cells.

• Klíčová slova
• ARF family, GTPases, eukaryotic cell, evolution, last eukaryotic common ancestor, posttranslational modifications,
• MeSH
• Eukaryota * genetika   MeSH
• eukaryotické buňky MeSH
• fylogeneze MeSH
• genom MeSH
• GTP-fosfohydrolasy * genetika   MeSH
• molekulární evoluce MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• GTP-fosfohydrolasy * MeSH

BRCA1-associated protein (BAP1)-inactivated melanocytic tumor (BIMT) is a group of epithelioid melanocytic neoplasms characterized by the loss of function of BAP1, a tumor suppressor gene located on chromosome 3p21. They occur sporadically or in the setting of an autosomal-dominant cancer susceptibility syndrome that predisposes to the development of different internal malignancies. Most of these cutaneous lesions are associated with a BRAF-mutated melanocytic nevus and therefore are included in the group of combined nevi in the last WHO classification of skin tumors. Apart from a BRAF mutation, an NRAS mutation has been reported in rare cases, whereas in some lesions no driver mutation has been detected. Here, we report 2 cases of BIMTs with a BAP1 mutation and a RAF1 fusion. Both lesions proved to be BRAF and NRAS wild type and were associated with a conventional melanocytic nevus with dysplastic junctional features. We suggest that RAF1 fusions can represent an underlying driver genetic event in these cases. Our study extends the morphological and molecular spectrum in BIMTs.

• MeSH
• dítě MeSH
• dospělí MeSH
• fenotyp MeSH
• fúze genů * MeSH
• genetická predispozice k nemoci MeSH
• GTP-fosfohydrolasy genetika   MeSH
• imunohistochemie MeSH
• lidé MeSH
• membránové proteiny genetika   MeSH
• mutační analýza DNA MeSH
• nádorové biomarkery analýza   genetika   MeSH
• nádorové supresorové proteiny genetika   MeSH
• nádory kůže chemie   genetika   patologie   chirurgie   MeSH
• pigmentový névus chemie   genetika   patologie   chirurgie   MeSH
• protoonkogenní proteiny B-Raf genetika   MeSH
• protoonkogenní proteiny c-raf genetika   MeSH
• thiolesterasa ubikvitinu genetika   MeSH
• umlčování genů * MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• BAP1 protein, human MeSH Prohlížeč
• BRAF protein, human MeSH Prohlížeč
• GTP-fosfohydrolasy MeSH
• membránové proteiny MeSH
• nádorové biomarkery MeSH
• nádorové supresorové proteiny MeSH
• NRAS protein, human MeSH Prohlížeč
• protoonkogenní proteiny B-Raf MeSH
• protoonkogenní proteiny c-raf MeSH
• Raf1 protein, human MeSH Prohlížeč
• thiolesterasa ubikvitinu MeSH