Original Mutation Research Articles

Current mechanism of acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors and updated therapy strategies in human nonsmall cell lung cancer.

Lung cancer continues to be a major health problem and the most common cancer-related mortality worldwide with about 80%-85% patients suffering from nonsmall cell lung cancer (NSCLC). More than 80% of NSCLC cases are often diagnosed as advanced stage and harbor epidermal growth factor receptor (EGFR) activating mutation. Although great success in initial response to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) are found in EGFR-mutant NSCLC patients, acquired resistance usually occurs on the continuous treatment. Here, we provide an overview on the mechanism of acquired resistance to EGFR-TKIs in NSCLC therapy as well as current preclinical and clinical evidence of new therapy strategies and inhibitors in the treatment of NSCLC. Many studies have shown that original or induced T790M mutation, human EGFR 2 amplification, and activated secondary signaling such as MET amplification or phosphatidylinositol 3-kinase mutation can lead to acquired resistance to EGFR-TKIs. In addition, transformation from NSCLC to SCLC or conferred epithelial to mesenchymal transition has also been identified as mechanisms of acquired resistance to EGFR-TKIs. Increasing evidence has proven that non-coding RNA including long noncoding RNAs and microRNAs or new EGFR mutation is involved in acquired resistance. Preclinical and clinical Phase 1-3 evidence on combination drug therapy or new generation inhibitors with different tumor-targeting approaches have made those strategies the promising options for EGFR-TKI-resistant NSCLC therapy. This review aims to get deep insight into providing a state-of-the-art overview of the recent advances in the mechanisms of acquired resistance and new strategies for targeted cancer therapy in EGFR-TKI-resistant NSCLC.

Genetic analysis of rice mutants responsible for narrow leaf phenotype and reduced vein number.

Leaves are a major site for photosynthesis and a key determinant of plant architecture. Rice produces thin and slender leaves, which consist of the leaf blade and leaf sheath separated by the lamina joint. Two types of vasculature, the large and small vascular bundles, run in parallel, together with a strong structure, the midrib. In this paper, we examined the function of four genes that regulate the width of the leaf blade and the vein number: NARROW LEAF1 (NAL1), NAL2, NAL3 and NAL7. We backcrossed original mutants of these genes with the standard wild-type rice, Taichung 65. We then compared the effect of each mutation on similar genetic backgrounds and examined genetic interactions of these genes. The nal1 single mutation and the nal2 nal3 double mutation showed a severe effect on leaf width, resulting in very narrow leaves. Although vein number was also reduced in the nal1 and nal2 nal3 mutants, the small vein number was more strongly reduced than the large vein number. In contrast, the nal7 mutation showed a milder effect on leaf width and vein number, and both the large and small veins were similarly affected. Thus, the genes responsible for narrow leaf phenotype seem to play distinct roles. The nal7 mutation showed additive effects on both leaf width and vein number, when combined with the nal1 single or the nal2 nal3 double mutation. In addition, observations of inner tissues revealed that cell differentiation was partially compromised in the nal2 nal3 nal7 mutant, consistent with the severe reduction in leaf width in this triple mutant.

Tracking the Somatic Mutations in Azacitidine-Treated MDS Patients Documents Clonal Development and AZA Responsiveness

Abstract Introduction and hypothesis: Somatic gene mutations develop in ~78% Myelodysplastic syndrome (MDS) patients. MDS progresses into an unstable phase characterized by an accumulation of myeloblasts that is indicated for the DNA demethylation therapy with Azacitidine (AZA). To understand whether AZA is capable to eliminate tumor cells and whether a mutation pattern responds to AZA we herein tracked mutations in the bone marrow (BM) during AZA treatment. Two scenarios were postulated: 1) AZA treatment will/ will not eliminate clones characterized by specific mutations and this will relate to the clinical outcome, 2) during clinical progression on the AZA treatment either the new mutations will develop or the original mutations will be detected. Patients: 40 int-2/high-risk MDS patients (176 samples, median age 70, 22F/18M) indicated for AZA (75mg/m2, 5+2+2) were sequenced. The MDS subtypes included RAEB1, RAEB2, and MDS/AML. Half of the patients progressed from the 5q-syndrome. Libraries were prepared using TruSight DNA amplicon kit. Set of 54 myeloid genes (associated with MDS or AML) were sequenced by Illumina platform HiSeq 2500 with depth >100 per mutation, mutation should be heterozygous, non-synonymous, exonic with frequency >10%. As controls: 4 normal BM samples and 2 cord bloods (also applied for data filtering) and MOLM-13 cell line carrying FLT3-ITD mutation were used. Additional controls included CD3+ T cells isolated from the patient BM samples. Results: 70% of patients were informative for at least one somatic mutation with high impact on the amino acid sequence. Mutations in TET2 (in 13%) were overall the most frequent before AZA was started (followed by mutations in BCOR, RUNX1, STAG2, and NOTCH genes). In 36% of informative patients, the mutation pattern developed on AZA. Interestingly, the most frequent mutations after AZA were in ASXL1 in 10% (followed by mutations in TET2, BCOR, and CUX1 genes). While mutations in ASXL1 and NOTCH genes developped only in the non-5q derived patients, the mutations in RUNX1 developped only in the 5q-derived higher-risk MDS patients. Average number of mutations before AZA and on AZA was 2 per patient. Complete elimination of the mutation pattern was noted in 57% of informative patients during the first 8 months of AZA treatment and this was associated with the therapy responsiveness (PR or CR). In contrast in 7% of informative patients the mutation pattern remained the same and this was associated with the stable disease (leading to progression). Upon progression on the AZA treatment we have observed appearance of the new mutations in 73% of informative patients. 20% of patients progressing after 14 cycles of AZA were non-informative which suggests that these patients may carry mutations in genes not included within the tested set. We have assessed overall survival (OS) according to the mutation status in the following subgroups: non-informative patients, informative patients with mutations that were eliminated by AZA, and those retaining or gaining the mutations upon AZA. The group that developed or retained mutations during first 4-8 cycles of AZA displayed significantly lower OS (p=0.004, median OS:11 months) compared to either non-informative patients (26 months) or those where AZA completely eliminated the mutation pattern (28 months). Conclusions: Tracking the mutations in MDS patients during AZA therapy provides opportunity to detect clonal development in 2/3 of the MDS patients and relate these data to the clinical outcome. Moreover, progression on AZA therapy is usually associated with the development of a new mutation pattern and this coincides with the significantly lower OS. Grants: GACR P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/1. Disclosures Stopka: Celgene: Research Funding.

Single Cell Analysis Proves the Coexistence of NOTCH1 and TP53 Mutations within the Same Cancer Cells in Patients with Chronic Lymphocytic Leukemia

IntroductionMutations in NOTCH1 and especially TP53 genes represent potent drivers of chronic lymphocytic leukemia (CLL) progression and chemo-refractoriness. Although a coexistence of these mutations was reported in CLL, a molecular basis of this phenomenon has not been described yet. To clarify this issue, we performed a detailed analysis of CLL patients with parallel NOTCH1 and TP53 mutations including single cancer cell examination.MethodsTP53 mutations were determined based on FASAY analysis coupled with direct sequencing. In a collected cohort of 111 TP53 -mutated patients a presence of hot spot c.7544_7545delCT NOTCH1 mutation was assessed using direct gDNA sequencing. In NOTCH1 -TP53 -mutated patients with available material, the mutations' coexistence was tested in single flow-sorted CD19+ cells (cancer cell proportion > 80 %) using multiplex PCR followed by direct sequencing.ResultsThe NOTCH1 mutation was detected in 19/111 (17 %) of the TP53 -mutated patients. Eleven of the NOTCH1-TP53 -mutated patients carried single TP53 mutation; multiple TP53 mutations were detected in 8 of them. Based on gDNA sequencing, the NOTCH1 and TP53 mutation coexistence in the same cancer cells was evident in 4/19 of the NOTCH1-TP53-mutated patients, as at least one of the gene mutations occurred in 100 % of the DNA. In the remaining 15 NOTCH1-TP53 -mutated patients the clonal composition was not possible to assess using sequencing data only and therefore a single cell analysis was performed in 8 of them with available material. Remarkably, irrespective of the mutation proportion, in all of these patients the NOTCH1 mutation was always present together with at least one of the detected TP53 mutations.Considering both the DNA sequencing and single cell analysis data, the 12 patientswith proven NOTCH1-TP53 mutation coexistence might be stratified into three groups with different clonal composition: i) patients with NOTCH1 and single TP53 mutations showing a comparable mutation proportion (n = 3), in which both gene mutations were always detected in the same cells and never occurred separately; ii) patients with either NOTCH1 or TP53 mutation predominance (n = 6), in which the predominant mutation was present separately as well as in combination with the coexisting mutation(s) in individual cells; iii) patients with NOTCH1 and multiple TP53 mutations showing different mutation proportion (n = 3), in which NOTCH1 mutation was present together with one of the detected TP53 mutations in the same cells, while the other TP53 mutations occurred separately.In two of the NOTCH1-TP53 -mutated patients who received intensive chemo-immunotherapy, the consecutive samples were available for single cell analysis. In one of these patients only single TP53 mutation was detected at first time point. In relapse after rituximab-dexamethasone treatment the clone carrying the original TP53 mutation expanded in parallel with another NOTCH1-TP53-mutated clone. Different situation was noticed in the second patient, in which the NOTCH1-TP53-mutated clone detected at first time point diminished after alemtuzumab treatment, while another TP53-mutated-NOTCH1-wild-type clone expanded in relapse.ConclusionWe have shown that in NOTCH1-TP53 -mutated patients the mutations often coexist in the same CLL cells. These patients exhibit a considerable clonal heterogeneity that may be further influenced by chemo-immunotherapy.This study was supported by IGA NT/13493 and NT/13519, MUNI/A/1180/2014, CZ.1.05/1.1.00/02.0068. DisclosuresMayer:Janssen: Research Funding.

Abstract A105: Credentialing the concept of “co-clinical trials”: Utility of lung cancer PDX models derived from patients on AZD9291 clinical trials

Abstract Background: The EGFR T790M mutation is the most common mechanism of acquired drug resistance to currently approved EGFR inhibitors gefitinib, erlotinib, and afatinib. AZD9291 is a mutant-selective EGFR inhibitor effective against both EGFR activating and T790M mutations while sparing wild type EGFR. AZD9291 is highly active in patients with lung cancer with the EGFR T790M mutation, with a response rate of 61% and progression free survival of ∼10 months. However, as resistance to AZD9291 is beginning to emerge, we aimed to develop patient derived xenografts (PDXs) using pre-treatment biopsies obtained from patients with acquired resistance to first-line EGFR inhibitors enrolling on clinical trials with AZD9291. These clinically annotated PDX models allow direct correlation with the clinical efficacy of AZD9291 in patients and may be useful in studying mechanisms of acquired resistance to AZD9291 and refine strategies for treatments. Methods: Pre-AZD9291 treatment tumor biopsies (core needle biopsies or pleural effusions) were implanted into the flank or sub-renal capsule of NSG mice. Tumors were serially passed in NSG mice for up to 3 generations. Successfully established models were expanded and treated with AZD9291. The efficacy in the PDX model was compared to the clinical efficacy of AZD9291 in the patient from whom the model was derived. Results: 33 patients underwent a pre-AZD9291 treatment biopsy (26 core needle; 7 pleural effusions). 26/33 patients enrolled in the AURA AZD9291 clinical trial for patients with acquired resistance to first-line EGFR inhibitors. From these patients, 10 PDX models have been successfully developed so far and confirmed by ddPCR to maintain fidelity to the original patient tumor's EGFR mutation status. These models include 6 with EGFR T790M and 4 with EGFR non-T790M mechanisms of resistance to erlotinib. Among the 10 patients used to develop PDX models, the best clinical response to AZD9291 included 5 partial responses, 3 progressive diseases, 1 stable disease and 1 acquired resistance. A subset of these PDX models were further tested for their sensitivity to AZD9291 and the data was consistent with the clinical responses of the patients. Two models, DFCI 243 and DFCI 217 (both with EGFR T790M; both patients with PR > 9 months) were treated with gefitinib (6.25mg/kg) or AZD9291 (25 mg/kg). Both models were confirmed to maintain EGFR T790M and had a dramatic response to AZD9291 but not to gefitinib treatment. However, tumors rapidly regrew upon cessation of AZD9291 treatment. DFCI 306 PDX model established from a patient with acquired resistance to monotherapy with AZD9291, showed AZD9291 resistance in vivo. AZD9291 treatment of DFCI 284, a model with EGFR T790M derived from a patient with primary resistance to AZD9291, is underway. Conclusion: We have developed PDXs from patients with erlotinib resistance who were treated with AZD9291. The PDX platform is currently being utilized in studies to refine strategies to: a) improve durability of responses to AZD9291 in EGFR T790M mutation positive patients and b) identify combinations in EGFR T790M mutation positive or negative patients who have de novo or acquired resistance to AZD9291. Citation Format: Sangeetha S. Palakurthi, Man Xu, Amanda Redig, Michael Dills, Prafulla Gokhale, Jihyun Choi, Atsuko Ogino, Yanan Kuang, Nora Feeney, Cloud Paweletz, Paul Kirschmeier, Jessie English, Darren Cross, Pasi A. Janne. Credentialing the concept of “co-clinical trials”: Utility of lung cancer PDX models derived from patients on AZD9291 clinical trials. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A105.

Application of experimental mutagenesis in plant breeding

The aim and tasks of the study. The purpose of the review is to assess the depth of studies and to overview subjects that are insufficiently explored in this field. Material and methods . Experiments on experimental mutagenesis of sunflower and amaranth were laid out in the experimental fields of the Plant Production Institute nd. a V. Ya. Yuriev and the Kharkiv National Agrarian University nd. a V.V. Dokuchaev in 2014. The source material in the sunflower studies was 12 self-pollinated lines bred at the Plant Production Institute nd. a V.Ya. Yuriev of NAAS, which were pretreated with chemical supermutagen solution (at the concentrations of 0.01% and 0.05%) and 12 self-pollinated lines, gamma-irradiated (120 and 150 Gray). The aim was to study effects nitrosoethylurea (NEU), dimethyl sulfate (DMS) and gamma-rays as a function of solution concentration, dose and exposure. The control was seeds watered for 18 hours. The source material in the amaranth ( Amaranthus hypohondriacus ) studies was three varieties – Sem, Kharkovskiyv 1 and Studencheskiy. In order to obtain economically valuable forms of amaranth, seeds were treated with physical mutagens (gamma irradiation). The source of radiation was Co 60 ; the doses were 10 Gy, 15 Gy and 30 Gy. In addition, seeds were irradiated with the high doses of 400 Gy and 700 Gy to determine the lethal dose for amaranth. Results and discussion . The study will establish peculiarities of nitrosoethylurea (NEU), dimethyl sulfate (DMS) and gamma-ray action on sunflower seeds and plants, depending on solution concentration, mutagen dose and exposure. The final study results will be source forms of sunflower with new features and properties that are valuable for breeding as well as economically valuable forms of amaranth. Conclusions . One method that enables obtaining source material, which would be diverse by many traits, in the short term is induced mutagenesis. Experimental mutagenesis is used for various purposes: obtainment of macro- and micromutations of qualitative and quantitative traits for selection or for direct practical use, negotiation of incapacity for distant hybridization, influence on crossingover in hybrids, suppression of the self-incompatibility reaction in cross-pollinated plants, induction of polyploidy, etc. At present, the research is focused on raising the efficiency of induction of original and breeding-valuable mutants as well as on the creation of fundamentally new sources of high productivity, yield capacity and resistance to biological and abiotic factors.

Enhanced survival of Drosophila Akt1 hypomorphs during amino-acid starvation requires foxo.

Disordered eating includes any pattern of irregular eating that may lead to either extreme weight loss or obesity. The conserved insulin receptor signalling pathway acts to regulate energy balance and nutrient intake, and its central component Akt1 and endpoint effector foxo are pivotal for survival during nutritional stress. Recently generated Akt1 hypomorphic mutant lines exhibit a moderate decrease in lifespan when aged upon standard media, yet show a considerable increase in survival upon amino-acid starvation media. While the loss of foxo function significantly reduces the survival response to amino-acid starvation, a combination of these Akt1 hypomorphs and a null foxo mutation reveal a synergystic and severe reduction in lifespan upon standard media, and an epistatic relationship when undergoing amino-acid starvation. Evaluation of survivorship upon amino-acid starvation media of these double mutants indicate a phenotype similar to the original foxo mutant demonstrating the role of foxo in this Akt1 phenotype. These results indicate that the subtle manipulation of foxo through Akt1 can enhance survival during adverse nutrient conditions to model the ability of individuals to tolerate nutrient deprivation. Ultimately, we believe that a Drosophila model of disordered eating could generate new avenues to develop potential therapies for related human conditions.

GENO-12CLONAL EVOLUTION AND GENOMIC DIVERSITY IN A PANEL GLIOBLASTOMA TUMORS, NEUROSPHERE CULTURES AND ORTHOTOPIC XENOGRAFTS

Molecular heterogeneity in glioblastoma (GBM) tumors presents major challenges to effective treatments. Well characterized patient-derived xenograft models are critical to support the development of better therapies and biomarkers. We established neurosphere cultures (NS) from twelve freshly resected primary and one recurrent GBM tumor and derived orthotopic xenografts in triplicate. All samples were analyzed using exome, low pass whole genome, RNA sequencing and reverse phase protein array (RPPA). We observed that 85% of mutations present in the tumor samples were transmitted to the NS. A majority of mutations shared by both tumor and NS were captured by xenografts with high cellular frequencies suggesting clonal mutations. Few mutations not present in the tumor samples were detected at low frequency in the NPs and approximately 66% of these were transmitted to the xenografts. A median number of 14 mutations were acquired in xenografts relative to NS, suggesting that the brain environment promotes novel somatic events. Genomic profiling of four matching late passage NS revealed that 15 % of the mutations detected in their early passage progenitors were lost, but as many as 59 novel mutations were acquired in extended culture. Consensus hierarchical gene expression profile clustering revealed that 70% of the tumors formed one cluster, while the NP and matching xenografts clustered together in several groups, including one characterized by EGFR amplification and mutations, another encompassing all the proneural subtype. Consistent with these observations, GBM transcriptional subtypes were conserved between tumor and matched NS in 42% of cases, and between NS and xenografts in 92% of cases. Importantly, signaling pathways significantly altered among sample modalities were identified by RPPA and RNAseq analysis, with implications for drug studies. In a panel of GBMs representative of all subclasses, remarkable conservation of original clonal mutations and evidence for clonal evolution were observed in the matched tumor-NP-xenografts.

G.P.332 - Charcot–Marie–Tooth type 4B1 (MTMR2 gene): Confounding clinical presentation and report of 5 original mutations

G.P.332 - Charcot–Marie–Tooth type 4B1 (MTMR2 gene): Confounding clinical presentation and report of 5 original mutations

Abstract 4910: Sensitive, specific and highly multiplexed mutation detection for cancer management

Abstract Mutations in RAS, RAF, EGFR and PIK3CA genes are present in a number of cancers including colon, lung and breast cancers. Importantly, many of these mutations are predictive biomarkers of clinical outcomes for targeted therapies. This has led to a growing demand for detection of multiple mutations simultaneously. Multiplex formats allow a greater amount of information to be obtained from each sample. PASS primer technology can sensitively and specifically identify the presence of SNPs and somatic DNA changes (point mutations, deletions and insertions) with sensitivity up to 1 in 1,000 (0.1%). PASS primers selectively amplify nucleic acid variants and create amplicons that are markedly different from the parent sequence. The method is superior to ARMS PCR for multiplexing, particularly when mutations are present at the same, adjacent or nearby loci. PASS primers consist of two target specific regions separated by an insert sequence (IS) which is not complementary to the target. A long 5′ target-specific region anchors the primer and a short 3′ region targets the variant base/s thus directing highly specific binding and extension. During amplification, the IS sequences are incorporated into amplicons, introducing ≥10 base difference between the starting single base variant and the resulting amplicon. This reduces competition between primers during amplification and facilitates robust discrimination between amplicons during detection. MNAzyme® qPCR is a robust alternative to other probe based real-time qPCR protocols such as TaqMan® and Beacons. MNAzymes are bi-specific, catalytic oligonucleotide complexes which form in the present of target and cleave universal reporter probes. The bi-specificity, and the use of well-characterised universal probes sets (suitable for use with any group of targets), makes MNAzyme® qPCR more amenable to multiplexing. When combined with PASS, MNAzyme® qPCR easily discriminates between amplicons since MNAzymes are tailored to specifically detect both the distinct IS and the original mutation. PASS MNAzyme® qPCR has been used to facilitate multiplex detection of RAS, RAF, EGFR and PIK3CA mutations. As many as eight mutations located at codons 12 and 13 of RAS have been successfully detected in a single well using three channels on a standard PCR machine. The technology is even more powerful when combined with newer instruments with higher multiplex capabilities such as Biocartis’ Idylla automated platform. A multiplexed KRAS/BRAF assay, which detects 18 mutations, was evaluated using colon cancer and melanoma FFPE samples. The results showed >96% concordance with sequencing and significantly specificity compared to the singleplex Therascreen KRAS test. Since PASS MNAzyme® qPCR affords greater multiplex capacity, along with high specificity and sensitivity, it provides a superior tool for ascertaining mutations from tumour tissues and is particularly well suited for use with liquid biopsies. Note: This abstract was not presented at the meeting. Citation Format: Lit Yeen Tan, Elisa Mokany, Samantha Walker, Tina Lonergan, Alison Todd. Sensitive, specific and highly multiplexed mutation detection for cancer management. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4910. doi:10.1158/1538-7445.AM2015-4910

Genetic shift in local rice populations during rice breeding programs in the northern limit of rice cultivation in the world.

The rapid accumulation of pre-existing mutations may play major roles in the establishment and shaping of adaptability for local regions in current rice breeding programs. The cultivated rice, Oryza sativa L., which originated from tropical regions, is now grown worldwide due to the concerted efforts of breeding programs. However, the process of establishing local populations and their origins remain unclear. In the present study, we characterized DNA polymorphisms in the rice variety KITAAKE from Hokkaido, one of the northern limits of rice cultivation in the world. Indel polymorphisms were attributed to transposable element-like insertions, tandem duplications, and non-TE deletions as the original mutation events in the NIPPONBARE and KITAAKE genomes. The allele frequencies of the KITAAKE alleles markedly shifted to the current variety types among the local population from Hokkaido in the last two decades. The KITAAKE alleles widely distributed throughout wild rice and cultivated rice over the world. These have accumulated in the local population from Hokkaido via Japanese landraces as the ancestral population of Hokkaido. These results strongly suggested that combinations of pre-existing mutations played a role in the establishment of adaptability. This approach using the re-sequencing of local varieties in unique environmental conditions will be useful as a genetic resource in plant breeding programs in local regions.

Genetic Screen for Suppressors of Y east Coq8 Mutants

Coenzyme Q (Q) is a lipid electron and proton carrier in the electron transport chain. Q functions in mitochondrial respiratory chain and serves as a lipophilic antioxidant. In yeast Saccharomyces cerevisiae, eleven genes designated as COQ1 through COQ9, YAH1 and ARH1, have been identified as being required for coenzyme Q biosynthesis. One of these eleven genes, COQ8, encodes Coq8 and previous studies suggest that Coq8 may function as a kinase required for Q biosynthesis. However, no experiments have yet shown any explicit kinase activity of Coq8 nor have phosphorylated forms of the Coq8 polypeptide been detected. Our goal in this project was to isolate coq8-3 revertants that can correct the Q biosynthetic defect and to perform genetic screens to identify novel protein suppressors present in the coq8-3 mutant. Both coq8-3 and coq8 null mutant lack Q6 and neither can grow on rich medium containing glycerol (YPG), a nonfermentable carbon source. Our results show that after 14 days of incubation, small yeast revertants were observed among the coq8-3 mutants plated on YPG plates. These revertants retain the original mutation present in the coq8-3 parent strain, but can grow in medium containing nonfermentable carbon sources. Ten coq8-3 revertants will further be analyzed and we hope to determine whether these revertants contain potential suppressor genes. It is our goal to identify any compensatory mutations that might help characterize Coq8 function. By understanding the kinase function of Coq8, this study will help us elucidate the eukaryotic biosynthetic pathway of Q, which is central to understanding and unraveling human genetic diseases related to Q deficiencies This research was supported by NSF MCB-1330803 and by NIH GM55052.

Morphological mutations: lessons from the cockscomb.

Morphological mutations: lessons from the cockscomb.

A long and winding road to the RNA world.

A long and winding road to the RNA world.

Characterization of a potato activation-tagged mutant, nikku, and its partial revertant.

A potato mutant with a strong stress-response phenotype, and a partial mutant revertant, were characterized. Gene expression patterns and DNA cytosine methylation varied between these and wild-type, indicating a role for DNA cytosine methylation changes in the gene expression and visible phenotypes. Morphological and molecular studies were conducted to compare potato cv. Bintje, a Bintje activation-tagged mutant (nikku), and nikku revertant phenotype plants. Morphological studies revealed that nikku plants exhibited an extremely dwarf phenotype, had small hyponastic leaves, were rootless, and infrequently produced small tubers compared to wild-type Bintje. The overall phenotype was suggestive of a constitutive stress response, which was further supported by the greater expression level of several stress-responsive genes in nikku. Unlike the nikku mutant, the revertant exhibited near normal shoot elongation, larger leaves and consistent rooting. The reversion appeared partial, and was not the result of a loss of 35S enhancer copies from the original nikku mutant. Southern blot analyses indicated the presence of a single T-DNA insertion on chromosome 12 in the mutant. Gene expression studies comparing Bintje, nikku and revertant phenotype plants indicated transcriptional activation/repression of several genes flanking both sides of the insertion in the mutant, suggesting that activation tagging had pleiotropic effects in nikku. In contrast, gene expression levels for many, but not all, of the same genes in the revertant were similar to Bintje, indicating some reversion at the gene expression level as well. DNA methylation studies indicated differences in cytosine methylation status of the 35S enhancers between the nikku mutant and its revertant. In addition, global DNA cytosine methylation varied between Bintje, the nikku mutant and the revertant, suggesting involvement in gene expression changes, as well as mutant phenotype.

Single site suppressors of a fission yeast temperature-sensitive mutant in cdc48 identified by whole genome sequencing.

The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors.

Hybrid evolutionary identification of output-error state-space models

A hybrid optimization method for the identification of state-space models is presented in this study. Hybridization is succeeded by combining the advantages of deterministic and stochastic algorithms in a superior scheme that promises faster convergence rate and reliability in the search for the global optimum. The proposed hybrid algorithm is developed by replacing the original stochastic mutation operator of Evolution Strategies (ES) by the Levenberg-Marquardt (LM) quasi-Newton algorithm. This substitution results in a scheme where the entire population cloud is involved in the search for the global optimum, while single individuals are involved in the local search, undertaken by the LM method. The novel hybrid identification framework is assessed through the Monte Carlo analysis of a simulated system and an experimental case study on a shear frame structure. Comparisons to subspace identification, as well as to conventional, self-adaptive ES provide significant indication of superior performance.

Clinical Outcome Following Change of Tyrosine Kinase Inhibitor (TKI) According to the Detection of an ABL Kinase Mutation

IntroductionPoint mutations in the kinase domain of BCR-ABL are the most frequent mechanism of acquired imatinib resistance in patients with chronic myeloid leukemia (CML). Mutation analysis is recommended to guide the selection of appropriate second line therapy in patients with imatinib failure, since some frequently occurring mutations confer clinical resistance to nilotinib and/or dasatinib. To date, more than 80 point mutations have been described following imatinib exposure, but mutations at 7 sites (G250, Y253, E255, T315, M351, F359, and H396) comprise approximately 60% of mutations reported in large series. We retrospectively analysed the impact of 83 ABL kinase mutations (P-loop mutations = 28, T315I mutation = 12 and other mutations = 43) arising in 65 chronic phase (CP) CML patients with imatinib failure. The aim of this study was to define the clinical characteristics of these patients, and to assess their outcome following introduction of second line agents.MethodsBetween July 2002 and August 2013, 123 CML patients were found to have ABL kinase mutations in our centre. Patients presenting in blast crisis (BC) or accelerated phase (AP), and those who did not require change in therapy (including patients who required imatinib dose escalation) following detection of an ABL kinase mutation were excluded from the analysis. Sixty-five patients in CP who had imatinib failure and detectable ABL kinase mutation, and who required change in therapy were evaluated. Definitions of CML phases, treatment responses and failures were as per definitions of the European LeukaemiaNet. Direct sequencing method was used to detect a range of mutations within the tyrosine kinase domain at the level of ~20% sensitivity and pyrosequencing to detect specific mutations with a sensitivity of ~5%.ResultsEighty-three ABL kinase mutations were detected in 65 CP patients at the time of imatinib failure with 35% of patients (23 of 65) harbouring P-loop mutations (including M244V), 18% (12 of 65) with T315I mutation and 46% (30 of 65) with other mutations (catalytic domain, imatinib binding site, activation loop and C-terminal). Composite mutations were present in 10 patients (15%), with 2 patients harbouring both P-loop and T315I mutations. Median time on imatinib therapy was 29.5 months (range, 2-144 months). At the time of mutation detection, 20% of patients (13 of 65) were in CCyR, 54% (35 of 65) in CP, 17% (11 of 65) in AP and 9% (6 of 65) had progressed to BC. Median time from CML diagnosis to mutation detection was 21 months for patients with T315I mutation, 45.5 months for P-loop mutations and 48 months for other mutations. Following mutation detection, patients in CP and AP were treated on second-line agents with dasatinib, nilotinib, bosutinib or ponatinib (based on sensitivity of ABL kinase mutants to ABL kinase inhibitors), and those in BC were treated with chemotherapy +/- TKI. Thirteen patients (20%) underwent allogeneic stem cell transplantation (SCT) for disease control (P-loop = 7, T315I = 5, other = 1).Following the change in TKI therapy, the best response was CMR or MMR in 54% of patients (35 of 65), CCyR in 12% (8 of 65), MCyR in 3% (2 of 65) and CHR in 26% (17 of 65). 5% (3 of 65) progressed to BC. In those who only achieved CHR, MCyR or developed progressive disease despite change in TKI, 6 out of 22 patients were found to have additional mutations during their treatment course, including 5 patients with re-emergence of their original mutation. After a median follow-up of 60 months (range, 3-137 months) from detection of mutation, 12 of the 65 patients (18%) have died, including 6 of 23 (26%) with P-loop mutations, 3 of 12 (25%) with T315I mutation, and 3 of 30 (10%) with other mutations. One patient who died had composite P-loop and T315I mutations. Median overall survival was 250 months for patients harbouring P-loop mutations (P=0.37) and not reached for T315I and other mutations.ConclusionWith longer follow-up and the availability of second and third generation TKIs, we have demonstrated that most clinically relevant ABL kinase mutations respond to change in TKI therapy following imatinib failure, with the majority of patients achieving durable cytogenetic and molecular response. This study emphasizes the importance of early detection and characterization of ABL kinase mutations in imatinib resistant patients in order to identify those patients who may benefit from alternative TKI therapy or stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Grouping evolution strategies: An effective approach for grouping problems

Many combinatorial optimization problems include a grouping (or assignment) phase wherein a set of items are partitioned into disjoint groups or sets. Introduced in 1994, the grouping genetic algorithm (GGA) is the most established heuristic for grouping problems which exploits the structural information along with the grouping nature of these problems to steer the search process. The aim of this paper is to evaluate the grouping version of the classic evolution strategies (ES) which originally maintain the well-known Gaussian mutation, recombination and selection operators for optimizing non-linear real-valued functions. Introducing the grouping evolution strategies (GES) to optimize the grouping problems that are intrinsically discrete, requests for developing a new mutation operator which works with groups of items rather than scalars and is respondent to the structure of grouping problems. As a source of variation, GES employs a mutation operator which shares a same rationale with the original ES mutation in the way that it works in continuous space while the consequences are used in discrete search space. A two phase heuristic procedure is developed to generate a complete feasible solution from the output of the mutation process. An extensive comparative study is conducted to evaluate the performance of GES versus GGA and GPSO (a recently proposed grouping particle swarm optimization algorithm) on test problem instances of the single batch-processing machine scheduling problem and the bin-packing problem. While these problems share exactly a same grouping structure and the performance of GES on both problems is reliable, switching from one problem to another deteriorates the performance of GGA. Though such a deficiency is not observed in the performance of GPSO, it is still inferior to GES on the single batch-processing machine scheduling test problem instances. Beside such empirical outcomes, the paper conveys a number of core strengths that the design of GES supports them but the design of GGA does not address them.

A previously uncharacterized tetratricopeptide-repeat-containing protein is involved in cell envelope function in Rhizobium leguminosarum.

Rhizobium leguminosarum is a soil bacterium that is an intracellular symbiont of leguminous plants through the formation of nitrogen-fixing root nodules. Due to the changing environments that rhizobia encounter, the cell is often faced with a variety of cell altering stressors that can compromise the cell envelope integrity. A previously uncharacterized operon (RL3499-RL3502) has been linked to proper cell envelope function, and mutants display pleiotropic phenotypes including an inability to grow on peptide-rich media. In order to identify functional partners to the operon, suppressor mutants capable of growth on complex, peptide-rich media were isolated. A suppressor mutant of a non-polar mutation to RL3500 was chosen for further characterization. Transposon mutagenesis, screening for loss of the suppressor phenotype, led to the identification of a Tn5 insertion in an uncharacterized tetratricopeptide-repeat-containing protein RL0936. Furthermore, RL0936 had a 3.5-fold increase in gene expression in the suppressor strain when compared with the WT and a 1.5-fold increase in the original RL3500 mutant. Mutation of RL0936 decreased desiccation tolerance and lowered the ability to form biofilms when compared with the WT strain. This work has identified a potential interaction between RL0936 and the RL3499-RL3502 operon that is involved in cell envelope development in R. leguminosarum, and has described phenotypic activities to a previously uncharacterized conserved hypothetical gene.