Acquisition Of Mutations In Genes Research Articles

Molecular and Functional Key Features and Oncogenic Drivers in Thymic Carcinomas.

Thymic epithelial tumors, comprising thymic carcinomas and thymomas, are rare neoplasms. They differ in histology, prognosis, and association with autoimmune diseases such as myasthenia gravis. Thymomas, but not thymic carcinomas, often harbor GTF2I mutations. Mutations of CDKN2A, TP53, and CDKN2B are the most common thymic carcinomas. The acquisition of mutations in genes that control chromatin modifications and epigenetic regulation occurs in the advanced stages of thymic carcinomas. Anti-angiogenic drugs and immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown promising results for the treatment of unresectable tumors. Since thymic carcinomas are frankly aggressive tumors, this report presents insights into their oncogenic drivers, categorized under the established hallmarks of cancer.

How CRISPR Is Revolutionizing the Generation of New Models for Cancer Research.

Cancers arise through acquisition of mutations in genes that regulate core biological processes like cell proliferation and cell death. Decades of cancer research have led to the identification of genes and mutations causally involved in disease development and evolution, yet defining their precise function across different cancer types and how they influence therapy responses has been challenging. Mouse models have helped define the in vivo function of cancer-associated alterations, and genome-editing approaches using CRISPR have dramatically accelerated the pace at which these models are developed and studied. Here, we highlight how CRISPR technologies have impacted the development and use of mouse models for cancer research and discuss the many ways in which these rapidly evolving platforms will continue to transform our understanding of this disease.

Genomic Abnormalities as Biomarkers and Therapeutic Targets in Acute Myeloid Leukemia.

Simple SummaryAML is a heterogenous malignancy with a variety of underlying genomic abnormalities. Some of the genetic aberrations in AML have led to the development of specific inhibitors which were approved by the Food and Drug Administration (FDA) and are currently used to treat eligible patients. In this review, we describe five gene mutations for which approved inhibitors have been developed, the response of AML patients to these inhibitors, and the known mechanism(s) of resistance. This review also highlights the significance of developing function-based screens for target discovery in the era of personalized medicine.Acute myeloid leukemia (AML) is a highly heterogeneous malignancy characterized by the clonal expansion of myeloid stem and progenitor cells in the bone marrow, peripheral blood, and other tissues. AML results from the acquisition of gene mutations or chromosomal abnormalities that induce proliferation or block differentiation of hematopoietic progenitors. A combination of cytogenetic profiling and gene mutation analyses are essential for the proper diagnosis, classification, prognosis, and treatment of AML. In the present review, we provide a summary of genomic abnormalities in AML that have emerged as both markers of disease and therapeutic targets. We discuss the abnormalities of RARA, FLT3, BCL2, IDH1, and IDH2, their significance as therapeutic targets in AML, and how various mechanisms cause resistance to the currently FDA-approved inhibitors. We also discuss the limitations of current genomic approaches for producing a comprehensive picture of the activated signaling pathways at diagnosis or at relapse in AML patients, and how innovative technologies combining genomic and functional methods will improve the discovery of novel therapeutic targets in AML. The ultimate goal is to optimize a personalized medicine approach for AML patients and possibly those with other types of cancers.

Single-Cell Analysis Based Dissection of Clonality in Myelofibrosis

Introduction Large-scale sequencing studies have unraveled the mutational landscape of myelofibrosis (MF), demonstrating clonal heterogeneity and importance of genetically defined subgroups in disease prognosis and progression. In order to elucidate the genetics of MF progression and its molecular drivers during JAK inhibition therapy, we performed in-depth genetic studies on longitudinal blood samples from 15 MF patients covering a disease span of 3 to 5 years after initiation of ruxolitinib. Methods Sequential samples from 15 MF patients (PMF n=8; post-ET/PV-MF n=7) accounting for a total of 42 time points representing 58.5 years of ruxolitinib treatment were investigated by whole-exome sequencing (WES). Additionally, we performed targeted deep sequencing of patient-specific mutations in flow-sorted cell fractions to study clonal repartition within the hematopoietic differentiation tree. Finally, we genotyped more than 5000 Lin-CD34+ progenitor cells using a single-cell multiplexed qPCR approach on a micro-fluidic platform (Fluidigm) to infer MF phylogeny. Results WES identified a median of 14 non-silent somatic mutations per patient at initiation of ruxolitinib treatment (=baseline WES; Figure 1A). When comparing mutations between first and last investigated time points, the majority of baseline mutations (162/201=81%) could be detected also at a later disease stage. A total of 39 mutations were lost and 80 new mutations were detected at the last time point. All patients showed at least one gained/ lost mutation in sequential samples. We noted frequent acquisition of mutations in genes of the RAS/RTK pathways in one third of patients. Two patients with a JAK2 V617F mutation achieved a molecular remission at a level of persisting residual disease of 1x10-3 with ruxolitinib therapy. In one of them, a total of 13 mutations were detected at baseline. In the second sample, taken three years later, a completely different set of mutations was identified and at the last time point, four years after initiation of therapy, none of the mutations were detected. This likely represents genetic drift during neutral evolution as a consequence of a rapid expansion after JAK inhibition. All other 13 patients showed only a modest - if any - decrease of 10-20% JAK2/CALR allele burden which was often accompanied with the expansion of JAK2/CALR-wildtype clones due to positive selection and/or freed clonal space under treatment. However, in some patients with durable response to ruxolitinib, we noted opposing dynamics of clones questioning a common origin. The three patients who progressed to leukemia showed a higher number of mutations at baseline and all of them acquired mutations in KRAS or NRAS over time. As one example, MPN18 harbored mutations in ASXL1, ETV6, and SRSF2 at baseline. Thereafter, and in addition to other driver genes (IDH2,KRAS) a second JAK2 Mutation at codon R867 was acquired, which has been reported to confer treatment resistance to JAK Inhibitors (Marty, Blood 2014). Mutation analysis in flow-sorted cell fractions showed a higher allelic mutation load in the myeloid compared to the lymphoid compartment with only few mutations being detected at low allele frequency in lymphocytes. Interestingly, some patients showed evidence of differential expansion among different myeloid cell lineages (Figure 1B). Next, we sorted 480 CD34+ single-cells per sample from 12 time points from 8 patients which allowed identification of subclones at ≥2% frequency based on priori power calculations. Sorting errors (e.g.cell doublets, empty wells)determined the mean cell sorting failure rate to be 12.5%. We employed a heuristic search algorithm to select a phylogenetic tree with Maximum Likelihood under a finite site model of evolution. Loss of heterozygosity (LOH) events were found in 7/8 patients and were not restricted to the JAK2 locus. In some patients, LOH of JAK2 occurred independently in two subclones, a phenomenon of convergent evolution (Figure 1C). We also noted cases with multiple 9pUPDs, of which one got selected during therapy. LOH events gave rise to both, a mutant homozygous but also reversion to a wildtype genotype. Conclusions Comprehensive serial genotyping of MF patients treated with ruxolitinib revealed heterogeneous patterns of clonal composition and evolution. Our data support LOH as a major determination factor for clonal diversification in MF. EM, KY, and MF contributed equally Figure 1 Disclosures Zenz: Abbvie: Consultancy, Honoraria, Other: Travel support; Roche: Consultancy, Other: Travel support; Janssen: Consultancy; Takeda: Consultancy; Gilead: Honoraria. Bullinger:Pfizer: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Abbvie: Honoraria; Bayer: Other: Financing of scientific research; Seattle Genetics: Honoraria; Sanofi: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria. Le Coutre:Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Ogawa:Kan Research Laboratory, Inc.: Consultancy; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding. Damm:Novartis: Research Funding; AbbVie: Other: Travel support.

Toward a genome-based treatment landscape for renal cell carcinoma.

Knowledge about molecular mechanisms driving development and progression of renal cell carcinoma has been elucidated by different studies. In few years we discovered a large difference between genomic landscapes of clear cell and non-clear cell carcinoma. Moreover, tumor heterogeneity and different acquisition of gene mutations during tumor progression are issues of particular interest. In this review we focalized our attention on principal genomic alterations identified among RCC subtypes. Acquired gene mutations may be an adaptive response to several external pressure including metabolic, treatment, genomic and immune-related external pressure. Thus we correlated and discussed principal genomic alterations adopted by tumor to escape from each external pressures. The aim of the present work is to summarize current knowledge about genomic alterations in RCC with special interest of treatment strategies tailored on the basis of disease mutations assessment.

Mechanisms of ciprofloxacin resistance in Pseudomonas aeruginosa: new approaches to an old problem.

The antibiotic ciprofloxacin is used extensively to treat a wide range of infections caused by the opportunistic pathogen Pseudomonas aeruginosa. Due to its extensive use, the proportion of ciprofloxacin-resistant P. aeruginosa isolates is rapidly increasing. Ciprofloxacin resistance can arise through the acquisition of mutations in genes encoding the target proteins of ciprofloxacin and regulators of efflux pumps, which leads to overexpression of these pumps. However, understanding of the basis of ciprofloxacin resistance is not yet complete. Recent advances using high-throughput screens and experimental evolution combined with whole-genome sequencing and protein analysis are enhancing our understanding of the genetic and biochemical mechanisms involved in ciprofloxacin resistance. Better insights into the mechanisms of ciprofloxacin resistance may facilitate the development of new or improved therapeutic regimes effective against P. aeruginosa. In this review we discuss the current understanding of the mechanisms of ciprofloxacin resistance and summarize the genetic basis of ciprofloxacin resistance in P. aeruginosa, in the context of current and future use of this antibiotic.

Aplastic anemia and clonal evolution: germline and somatic genetics

Clonal progression to myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) remains a dreaded complication for a subset of patients with bone marrow failure (BMF). Recognizing risk factors for the development of MDS or AML would inform individualized treatment decisions and identify patients who may benefit from early or upfront hematopoietic stem cell transplantation. Now that next-generation DNA sequencing is available in the clinical laboratory, research has focused on the implications of germline and somatic mutations for diagnosing and monitoring patients with BMF. Most germline genetic BMF disorders are characterized by a high propensity to develop MDS or AML. Recent studies of somatic variants in marrow failure revealed a high frequency of clonal hematopoiesis with the acquisition of mutations in genes associated with MDS or AML. The evaluation and implications of germline and somatic mutations for the development of clonal disorders in patients with BMF and challenges of clinical genetic testing will be summarized in this paper based on the reports from the 58th American Society of Hematology (ASH) Annual Meeting. Key words: Bone marrow failure; Anemia, aplastic; Myelodysplastic syndrome; Leukemia, myeloid, acute; Germline genetics; Somatic genetics; Clonal progression; Next generation sequencing; American Society of Hematology Annual Meeting

Aplastic anemia and clonal evolution: germ line and somatic genetics.

Clonal progression to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) remains a dreaded complication for a subset of patients with bone marrow failure (BMF). Recognizing risk factors for the development of MDS or AML would inform individualized treatment decisions and identify patients who may benefit from early or upfront hematopoietic stem cell transplantation. Now that next-generation DNA sequencing is available in the clinical laboratory, research has focused on the implications of germ line and somatic mutations for diagnosing and monitoring patients with BMF. Most germ line genetic BMF disorders are characterized by a high propensity to develop MDS or AML. Many affected patients lack the physical stigmata traditionally associated with the inherited marrow failure syndromes. Although any single inherited marrow failure disorder is rare, multiplexed genetic sequencing that allows simultaneous evaluation of marrow failure genes en masse demonstrated that, as a group, these inherited disorders compose a significant subset (5% to 10%) of patients with BMF. Early diagnosis of a germ line genetic marrow failure disorder allows individualized monitoring and tailored therapy. Recent studies of somatic variants in marrow failure revealed a high frequency of clonal hematopoiesis with the acquisition of mutations in genes associated with MDS or AML. Investigation of somatic mutations in marrow failure revealed important insights into the mechanisms promoting clonal disease but also raised additional questions. This review will focus on the evaluation and implications of germ line and somatic mutations for the development of clonal disorders in patients with BMF. Challenges and limitations of clinical genetic testing will be explored.

The Genomic Basis of Evolutionary Innovation in Pseudomonas aeruginosa.

Novel traits play a key role in evolution, but their origins remain poorly understood. Here we address this problem by using experimental evolution to study bacterial innovation in real time. We allowed 380 populations of Pseudomonas aeruginosa to adapt to 95 different carbon sources that challenged bacteria with either evolving novel metabolic traits or optimizing existing traits. Whole genome sequencing of more than 80 clones revealed profound differences in the genetic basis of innovation and optimization. Innovation was associated with the rapid acquisition of mutations in genes involved in transcription and metabolism. Mutations in pre-existing duplicate genes in the P. aeruginosa genome were common during innovation, but not optimization. These duplicate genes may have been acquired by P. aeruginosa due to either spontaneous gene amplification or horizontal gene transfer. High throughput phenotype assays revealed that novelty was associated with increased pleiotropic costs that are likely to constrain innovation. However, mutations in duplicate genes with close homologs in the P. aeruginosa genome were associated with low pleiotropic costs compared to mutations in duplicate genes with distant homologs in the P. aeruginosa genome, suggesting that functional redundancy between duplicates facilitates innovation by buffering pleiotropic costs.

Hypoxia-HIF-1α-C/EBPα/Runx1 signaling in leukemic cell differentiation

Acute myeloid leukemia (AML), a class of prevalent hematopoietic malignancies, is caused by the acquisition of gene mutations that confer deregulated proliferation, impaired differentiation and a survival advantage of hematopoietic progenitors. More recently, we reported that cobalt chloride (CoCl 2)/iron chelator desferrioxamine (DFO)-mimicked hypoxia or moderate hypoxia (2% and 3% O 2) can directly trigger differentiation of many subtypes of AML cells. Also, intermittent hypoxia significantly prolongs the survival of the transplanted leukemic mice with differentiation induction of leukemic cells. Additionally, these hypoxia-simulating agents selectively stimulate differentiation in acute promyelocytic leukemic cells induced by arsenic trioxide, an effective second-line drug for this unique type of leukemia. Based on this interesting evidence in vitro and in vivo, the ongoing investigations showed the role of hypoxia-inducible factor-1alpha (HIF-1α) protein through its non-transcriptional activity in myeloid cell differentiation, as evidenced by chemical interference, the conditional HIF-1α induction, the specific short hairpin RNAs (shRNAs) against HIF-1α and HIF-1β, an essential partner for transcription activity of HIF-1. Furthermore, HIF-1α and two hematopoietic transcription factors CCAAT/enhancer binding protein alpha (C/EBPα) and Runx1/AML1 interact directly with each other. Such interactions increase the transcriptional activities of C/EBPα and Runx1/AML1, while C/EBPα competes with HIF-1β for direct binding to HIF-1α protein, and significantly inhibits the DNA-binding ability of HIF-1. As a protein is rapidly responsive to all-trans retinoic acid (ATRA), a classical clinical differentiation-inducing drug for AML, HIF-1α also plays a role in ATRA-induced differentiation of leukemic cells.