Characterization and Clinical Significance of EIF1AX Mutations and Co-Mutations in Cytologically Indeterminate Thyroid Nodules: A 5-Year Retrospective Analysis.

OBJECTIVE: Mutations in the EIF1AX gene have been recently detected in a small percentage of benign and malignant thyroid lesions. We sought to investigate the prevalence and clinical significance of EIF1AX mutations and co-mutations in cytologically indeterminate thyroid nodules at our institution. MATERIALS AND METHODS: A 5-year retrospective analysis was performed on thyroid nodules with a cytologic diagnosis of Bethesda category III or IV, which had undergone testing by our in-house next generation sequencing panel. Surgically resected nodules with EIF1AX mutations were identified, and mutation type and presence of co-mutations were correlated with histopathologic diagnosis. RESULTS: 41/904 (4.5%) cases overall and 26/229 (11.4%) surgically resected nodules harbored an EIF1AX mutation. The most common histologic diagnoses were follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. 11/26 (42.3%) of nodules had isolated EIF1AX mutation. Comutations were found in RAS (12/26; 46.2%), TERT (5/26; 19.2%) and TP53 (2/26; 7.7%). EIF1AX mutation alone conferred a 36.4% risk of malignancy (ROM) and 54.5% ROM or noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), while the ROM was significantly higher in nodules with concurrent RAS (71.4%), TERT, TP53 and RAS+TERT (100%) mutations. CONCLUSION: EIF1AX mutations occur in benign and malignant follicular thyroid neoplasms. In our cohort, the majority of mutations occurred at the splice acceptor site between exons 5 and 6. Importantly, the coexistence of EIF1AX mutations with other driver pathogenic mutations in RAS, TERT and TP53 conferred a 100% ROM or NIFTP, indicating that such nodules require surgical removal

Lessons from Trypanosome TFIIH: Discovery of the Essential Roles of CRK9 in Gene Expression and of the Divergent Helicase Paralog XPB-R in Nucleotide Excision Repair

Eukaryotic TFIIH consists of a core of seven subunits, including the DNA helicase Xeroderma Pigmentosum B (XPB), and a cyclin-dependent kinase (CDK)-activating complex (CAK) that contains CDK7. XPB is crucial for DNA unwinding during transcription and nucleotide excision repair (NER) while the CAK complex phosphorylates RNA polymerase II (RNAPII) carboxy terminal domain (CTD), enabling the enzyme’s promoter clearance. Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are lethal human parasites, belonging to the early-diverged order Kinetoplastida. They transcribe genes polycistronically and require spliced leader (SL) trans splicing for the maturation of mRNAs from polycistronic precursors. SL RNA gene (SLRNA) transcription by RNAPII depends on trypanosome TFIIH complex which contains orthologs of all core subunits but lacks the CAK complex. Despite this, the trypanosome CTD is phosphorylated and essential for SLRNA transcription. Gene silencing of CDC2-related kinase 9 (CRK9) revealed its importance for parasite survival and CTD phosphorylation. Interestingly, this loss of phosphorylation did not cause a specific RNAPII transcription defect. Instead, CRK9 silencing blocked trans splicing and caused hypomethylation of SL RNA’s extensively modified cap. Sedimentation analysis of tandem affinity purified CRK9 revealed a putative tripartite complex including a novel L-type cyclin (CYC12), and a CRK9-associated protein (CRK9AP). Silencing CYC12 or CRK9AP recapitulated the defects observed upon CRK9 silencing, confirming that they functionally partner with CRK9 in vivo. CRK9AP depletion caused a rapid co-loss of CRK9 and CYC12, suggesting its role in complex assembly. This project identified CRK9 as crucial for parasite-specific gene expression. As a CDK, CRK9 is a promising drug target against trypanosomes which was validated in a mouse model. Another feature related to trypanosome TFIIH is the presence of two divergent paralogs of XPB in kinetoplastid genomes while only the larger XPB paralog consistently co-purified with TFIIH. Gene knockout of the second paralog, termed XPB-R showed that XPB-R is specialized in NER but dispensable for transcription. While XPB-R does not assemble into a TFIIH complex, reciprocal co-immunoprecipitations revealed an interaction with the p52 ortholog, a TFIIH component and known regulator of XPB in other systems, indicating that trypanosomes possess a TFIIH whose function is restricted to transcription and a XPB-R/p52 repair complex

Cyclin-Dependent Kinase CRK9, Required for Spliced Leader trans Splicing of Pre-mRNA in Trypanosomes, Functions in a Complex with a New L-Type Cyclin and a Kinetoplastid-Specific Protein.

In eukaryotes, cyclin-dependent kinases (CDKs) control the cell cycle and critical steps in gene expression. The lethal parasite Trypanosoma brucei, member of the phylogenetic order Kinetoplastida, possesses eleven CDKs which, due to high sequence divergence, were generically termed CDC2-related kinases (CRKs). While several CRKs have been implied in the cell cycle, CRK9 was the first trypanosome CDK shown to control the unusual mode of gene expression found in kinetoplastids. In these organisms, protein-coding genes are arranged in tandem arrays which are transcribed polycistronically. Individual mRNAs are processed from precursor RNA by spliced leader (SL) trans splicing and polyadenylation. CRK9 ablation was lethal in cultured trypanosomes, causing a block of trans splicing before the first transesterification step. Additionally, CRK9 silencing led to dephosphorylation of RNA polymerase II and to hypomethylation of the SL cap structure. Here, we tandem affinity-purified CRK9 and, among potential CRK9 substrates and modifying enzymes, discovered an unusual tripartite complex comprising CRK9, a new L-type cyclin (CYC12) and a protein, termed CRK9-associated protein (CRK9AP), that is only conserved among kinetoplastids. Silencing of either CYC12 or CRK9AP reproduced the effects of depleting CRK9, identifying these proteins as functional partners of CRK9 in vivo. While mammalian cyclin L binds to CDK11, the CRK9 complex deviates substantially from that of CDK11, requiring CRK9AP for efficient CRK9 complex formation and autophosphorylation in vitro. Interference with this unusual CDK rescued mice from lethal trypanosome infections, validating CRK9 as a potential chemotherapeutic target

Patient with Multiple Genetically Distinct Thyroid Nodules Including Papillary Thyroid Carcinoma Harboring Novel YWHAG-BRAF Fusion

Next-generation sequencing (NGS) analysis of thyroid samples aids in risk stratification of cytologically indeterminate nodules and contributes to our understanding of molecular mechanisms in thyroid neoplasia. Several genes, including BRAF, RAS, and EIF1AX, are known to play a role in thyroid tumorigenesis. Here we report a case of papillary thyroid carcinoma (PTC) in which a single lesion harbored a novel YWHAG-BRAF fusion and EIF1AX mutation and displayed mixed morphological findings. The patient is a 74-year-old female with multiple incidentally discovered thyroid nodules, two of which were sampled by ultrasound-guided fine needle aspiration (FNA). Cytologic diagnosis for both nodules was suspicious for follicular neoplasm (Bethesda Category IV). NGS testing of one nodule detected a novel in-frame YWHAG-BRAF fusion and a concurrent EIF1AX A113 splice mutation. The subsequent surgical resection specimen showed that this nodule exhibited two distinct morphologic patterns, conventional (classical) type and follicular variant (FV) of PTC, which were sharply demarcated and were found to harbor unique genetic alterations. Of note, this is the first report of BRAF activation through novel rearrangement with a gene encoding a 14-3-3 protein as a pathogenic factor, which underlines its significance both as a prognostic measurement and as a therapeutic target

CYC12 and CRK9AP are functional partners of CRK9.

<p>(A) Cumulative culture growth curves were obtained for <i>CYC12</i> and <i>CRK9AP</i> silencing in the absence and presence of doxycycline (dox), the gene knockdown-inducing compound. For each knockdown a representative growth curve is shown. (B) Analysis of total RNA prepared from non-induced cells and cells in which <i>CYC12</i> or <i>CRK9AP</i> were silenced for 1, 2 or 3 days. <i>CYC12</i> or <i>CRK9AP</i> mRNA as well as α tubulin and <i>RPB7</i> mRNA were analyzed by reverse transcription of oligo-dT and semi-quantitative PCR, whereas unspliced, pre-mRNA of α tubulin and <i>RPB7</i> were analyzed by reverse transcription of random hexamers and by PCR using an oligonucleotide upstream of the SL addition site. rRNA was visualized by ethidium bromide staining after separation in an agarose gel. SL RNA, U2 snRNA and the Y structure intermediate were detected by primer extension assays using a SL RNA and a U2 snRNA-specific primer in the same reactions. (C) Anti-RPB1 immunoblot analysis of whole-cell lysates prepared from <i>CRK9AP</i>-silenced cells. Detection of the similar-sized RNA pol I subunit RPA1 served as a loading control.</p

Validation of CRK9 as a drug target in the mouse.

<p>(A and B, top) Depiction of CRK9 mRNAs and the targeting dsRNA in two cell lines derived from the <i>T</i>. <i>brucei brucei</i> 427 smBF cell line which were used for mouse infection studies. The cell line on the left (A) harbored a construct for conditional expression of dsRNA that targets the 3^/ UTR of the <i>CRK9</i> mRNA. This line was further modified (B) by targeted integration of a plasmid into the endogenous <i>CRK9</i> locus that fused a functional HA tag sequence and the 3^/ UTR of <i>RPA1</i> to the 3^/ end of one <i>CRK9</i> allele, making the corresponding mRNA resistant to the RNAi response. As the survival graphs of infected mice show in the bottom panels, doxycycline treatment rescued every single mouse when CRK9 was depleted. This effect was completely abolished upon introduction of an RNAi-resistant <i>CRK9</i> gene into the same trypanosomes.</p

Cyclin CYC12 is an L-type cyclin.

<p>(A) Schematic drawing to scale of the human L1 and <i>T</i>. <i>brucei</i> CYC12 cyclins. The two cyclin folds (blue) are embedded in the CCL1 domain (green). The charged RS domain (red) was defined by a hydrophilicity Kyte & Doolittle blot score of < -2. Black lines indicate SR or RS dipeptides. Both cyclin domains of CYC12 are disrupted by insertions. (B) Phylogenetic tree, generated by the maximum likelihood algorithm and based on a multiple sequence alignment of the cyclin domains of human cyclins and of CYC12s from <i>T</i>. <i>brucei</i> (Tb), <i>T</i>. <i>cruzi</i> (Tc), <i>L</i>. <i>major</i> (Lm) and the bodonid <i>Bodo saltans</i> (Bs) (for accession numbers see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005498#ppat.1005498.s005" target="_blank">S5 Fig</a>). Cyclins involved in the cell cycle and in transcriptional control are indicated. Bootstrap values are indicated in percentages and were derived from 1000 replicates. The common branch of human cyclins L and kinetoplastid CYC12s is drawn in red.</p

CRK9 interacts and co-sediments with two unannotated proteins.

<p>(A) CRK9-PTP tandem affinity-purified material was sedimented through a 10–40% linear sucrose gradient by ultracentrifugation and fractionated into 20 aliquots from top to bottom. Note that pelleted proteins were resuspended in fraction 20 (20+P). Proteins from each fraction were separated by SDS-PAGE and stained with SYPRO Ruby. Protein bands were excised and identified by LC/MS/MS. Arrows point to the CYC12 and CRK9AP bands which co-sediment with CRK9 in fractions 9/10. The 35 kDa band with a peak in fractions 10/11 was found to be the putative ribosomal protein L5 (Tb927.9.15110/15150). (B) Kinase assay with materials from indicated fractions suggest autophosphorylation of CRK9. (C) Reciprocal co-IP assays of extracts prepared from a cell line in which CRK9 was exclusively PTP-tagged and an HA tag sequence was inserted at the 3’end of one CYC12 allele. The precipitate (P) was loaded at a fourfold excess relative to extract (Inp) and supernatant (S). Detection of the RNA pol II transcription factor TFIIB served as a negative precipitation control.</p

CRK9AP depletion results in rapid co-loss of CRK9 and CYC12.

<p>(A) Immunoblot of whole cell lysates derived from non-induced (n.i.) and <i>CRK9AP</i>-silenced PF trypanosomes. The arrow indicates the gene knockdown of <i>CRK9AP</i>. Detection of the class I transcription factor A subunit 6 (CITFA6) served as a loading control. (B) Corresponding semi-quantitative PCR analysis of cDNA that was obtained from the same cells by reverse transcription of total RNA using oligo-dT. Relative RNA amounts were determined by ethidium bromide–stained rRNA.</p

CRK9AP is essential for CRK9 enzyme assembly and autophosphorylation.

<p>(A) Schematic to scale of recombinant CRK9-PTP, CYC12^1-518-HA, and CRK9AP proteins that were expressed in wheat germ extract. PTP and HA tags are depicted as black boxes. (B) rCYC12^1-518-HA and rCRK9-PTP were pulled down from extract by anti-HA and IgG beads that bind to ProtA of the PTP tag, respectively. Pulldown and co-precipitation (asterisks) of CRK9 complex subunits were analyzed by immunoblotting with anti-ProtC (PTP tag), anti-HA and anti-CRK9AP antibodies, detecting the three proteins in extract (Inp), supernatant (S) and precipitate (P) which was loaded in six-fold excess to extract and supernatant. Negative control pulldowns (ctrl IP) were carried out with extract in which the target protein was not expressed. Note that IgG beads but not anti-HA beads reproducibly co-precipitated minor amounts of either rCYC12^1-518-HA and rCRK9AP in the control assays. (C) Kinase assay after IgG affinity chromatography and TEV protease release of rCRK9-P in the presence of all three complex components or with either CRK9AP or rCYC12^1-518-HA. In a negative control (neg ctrl), the assay was carried out without expression of trypanosome proteins and, in a positive control (end CRK9), CRK9 autophosphorylation was achieved by the endogenous CRK9 complex that was tandem affinity-purified from trypanosome extract. The labeled CRK9-P band is indicated on the right (autophosphorylation).</p