scholarly journals The likelihood of heterogeneity or additional mutation in KRAS or associated oncogenes to compromise targeting of oncogenic KRAS G12C

2017 ◽  
Author(s):  
Vincent L. Cannataro ◽  
Stephen G. Gaffney ◽  
Carly Stender ◽  
Zi-Ming Zhao ◽  
Mark Philips ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
De Novo ◽  
Point Mutations ◽  
Treatment Resistance ◽  
Braf V600e ◽  
Intratumor Heterogeneity ◽  
Resistance Mutations ◽  
Ras Genes ◽  
Fitness Advantage

AbstractActivating mutations in RAS genes are associated with approximately 20% of all human cancers. New targeted therapies show preclinical promise in inhibiting the KRAS G12C variant, however, concerns exist regarding the effectiveness of such therapies in vivo given the possibilities of existing intratumor heterogeneity or de novo mutation leading to treatment resistance. We performed deep sequencing of 27 KRAS G12 positive lung tumors and found no evidence of other oncogenic mutations within KRAS or within commonly mutated downstream genes that could confer resistance at the time of treatment. Furthermore, we estimate the de novo mutation rate in KRAS position 12 and in genes downstream of KRAS. We find that mutations that confer resistance are about as likely to occur downstream of KRAS as within KRAS. Moreover, we present an approach for estimation of the selection intensity for these point mutations that explains their high prevalence in tumors. Our approach predicts that BRAF V600E would provide the highest fitness advantage for de novo resistant subclones. Overall, our findings suggest that resistance to targeted therapy of KRAS G12C positive tumors is unlikely to be present at the time of treatment and, among the de novo mutations likely to confer resistance, mutations in BRAF, a gene with targeted inhibitors presently available, result in subclones with the highest fitness advantage.One Sentence SummaryMutations conferring resistance to KRAS G12C targeted therapy are unlikely to be present at the time of resection, and the likely mechanisms of evolved resistance are predicted be ones that are responsive to therapies that are in development or that are already available.

scholarly journals Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 146
Author(s):  
Angelo Pavesi ◽  
Fabio Romerio
Keyword(s):  
De Novo ◽  
Point Mutations ◽  
Selective Advantage ◽  
Genomic Region ◽  
Fine Tuning ◽  
Sequence Evolution ◽  
Viral Genomes ◽  
Stop Codons ◽  
Hiv 1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

scholarly journals Bacteriophage adaptation to a mammalian mucosa reveals a trans–domain evolutionary axis

2021 ◽  
Author(s):  
Wai Hoe Chin ◽  
Ciaren Kett ◽  
Oren Cooper ◽  
Deike Müseler ◽  
Yaqi Zhang ◽  
...  
Keyword(s):  
De Novo ◽  
Genetic Recombination ◽  
Evolutionary Relationship ◽  
Mammalian Host ◽  
Single Mutation ◽  
Phage Ecology ◽  
Fitness Advantage ◽  
Gut Mucosa

The majority of viruses within the human gut are obligate bacterial viruses known as bacteriophages (phages)1. Their bacteriotropism underscores the study of phage ecology in the gut, where they sustain top–down control2—4 and co–evolve5 with gut bacterial communities. Traditionally, these were investigated empirically via in vitro experimental evolution6—8 and more recently, in vivo models were adopted to account for gut niche effects4,9. Here, we probed beyond conventional phage–bacteria co–evolution to investigate the potential evolutionary interactions between phages and the mammalian ″host″. To capture the role of the mammalian host, we recapitulated a life–like mammalian gut mucosa using in vitro lab–on–a–chip devices (to wit, the gut–on–a–chip) and showed that the mucosal environment supports stable phage–bacteria co–existence. Next, we experimentally evolved phage populations within the gut–on–a–chip devices and discovered that phages adapt by de novo mutations and genetic recombination. We found that a single mutation in the phage capsid protein Hoc — known to facilitate phage adherence to mucus10 — caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan–binding phenotype provided the evolved mutant phage a competitive fitness advantage over their ancestral wildtype phage in the gut–on–a–chip mucosal environment. Collectively, our findings revealed that phages — in addition to their evolutionary relationship with bacteria — are also able to engage in evolution with the mammalian host.

scholarly journals Counteracting resistance to targeted therapy in melanoma by inhibiting discoidin domain receptors

2019 ◽  
Author(s):  
Margaux Sala ◽  
Nathalie Allain ◽  
Elodie Henriet ◽  
Arnaud Uguen ◽  
Sylvaine Di-Tommaso ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Map Kinase ◽  
Braf V600e ◽  
First Line ◽  
Discoidin Domain Receptors ◽  
Map Kinase Pathway ◽  
Kinase Pathway ◽  
Resistant Cells

AbstractAnti-BRAF plus an anti-MEK is currently used in first line for the management of patients presenting metastatic melanomas harboring the BRAF V600E mutation. However, the main issue during targeted therapy is the acquisition of cellular resistance in 80% of the patients, which is associated with an increased metastasis due to the hyperactivation of MAP kinase pathway. Previous reports have indicated that Discoidin Domain Receptors (DDRs) 1 and 2 can activate this pathway. To study the role of DDRs in melanoma cell resistance to targeted therapy, we first determined that DDRs are overexpressed in vemurafenib resistant cells compared to sensitive cells. We demonstrated that DDRs depletion or inactivation by DDRs inhibitors such as dasatinib or CR-13542 reduces tumor cell proliferation, due to a decrease of MAP kinase pathway activity in resistant cells. Finally, we confirmed these results in vivo in a xenograft mouse model and show that DDRs could be new therapeutic targets in resistant patients with metastatic melanoma. We propose that dasatinib could be a second-line treatment after the bi-therapy in resistant patients overexpressing DDRs.

scholarly journals Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687 ◽  
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

TTT-3002 Is Active Against Relapsed/Refractory AML Patient Bone Marrow With FLT3 TKI-Resistance Mutations At Residue D835

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3818-3818
Author(s):  
Hayley S Ma ◽  
Bao Nguyen ◽  
Mark Levis ◽  
Allen B Williams ◽  
Donald Small
Keyword(s):  
Drug Resistance ◽  
Clinical Trials ◽  
Ex Vivo ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Resistance Mutations ◽  
Patient Sample ◽  
Tki Resistance ◽  
Selection Of

Abstract Over 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FLT3, either internal tandem duplication (ITD) or point mutations (FLT3/PM). FLT3/ITD mutations in particular confer a poor prognosis, and thus several FLT3 tyrosine kinase inhibitors (TKIs) have been developed and are currently in clinical trials. However, many have failed due to insufficient achievement of FLT3 kinase inhibition in vivo. This is thought to be due to insufficient potency, reduced activity against FLT3/PM, and/or selection for resistance-conferring point mutations in FLT3/ITD. Therefore, the search for novel FLT3 TKIs that overcome some of these resistance mechanisms that result in persistent FLT3 activation is necessary to improve the cure rate for this disease. We have previously reported on TTT-3002, a novel TKI that possesses the most potent activity against FLT3 phosphorylation, with IC50s of 100-250pM. Here we characterize the activity of this compound against TKI-resistant AML. The phenomenon of drug resistance associated with selection of FLT3 point mutations is being observed with increasing frequency as higher levels of inhibition by FLT3 TKIs are achieved in clinical trials of FLT3/ITD AML patients. In order to predict the ability of TTT-3002 to successfully treat some of these resistance mutations, we used site-directed mutagenesis to generate a series of FLT3/ITD TKI resistant cell lines. We directly compared the activity of TTT-3002 to other FLT3 TKIs currently in clinical trials including CEP-701, sorafenib, AC220 and PKC412. TTT-3002 remained the most potent inhibitor of all six FLT3/ITD TKI resistance mutants screened, with viability IC50 values of less than 1nM (with the single exception of Ba/F3-G697R/ITD, IC50=11nM). It was thus able to effectively inhibit FLT3 with TKI resistance mutations against which other TKIs are ineffective. Therefore, this compound’s broad spectrum of activity against FLT3/ITD TKI resistance mutations may enable it to successfully treat FLT3/ITD AML patients who have become resistant to other FLT3 TKIs. To examine this directly, human AML samples were obtained from patients that developed resistance to sorafenib or AC220 while on clinical trials and presented with a dual D835/ITD mutation or D835 point mutation alone at the time of relapse. These blasts were evaluated for their ex vivo sensitivity to TTT-3002, sorafenib and AC220, with a diagnostic FLT3/ITD+ patient sample for comparison. Encouragingly, we observed significant effects on proliferation, induction of apoptosis, and cell signaling when cells were treated with TTT-3002. AC220 was moderately active against FLT3/ITD+ blasts and one patient sample with a D835/ITD mutation, and sorafenib was active against only the diagnostic FLT3/ITD sample, but none of the three relapse samples. Therefore, TTT-3002 maintains activity against human AML patient samples that are resistant in clinical trials to the FLT3 TKIs sorafenib and AC220. To account for the patterns of drug resistance observed for different FLT3 TKIs in our study, we modeled the positions of staurosporine-like (TTT-3002, CEP-701 and PKC412) and sorafenib-like inhibitors (sorafenib and AC220) bound to FLT3. Examination of amino-acid substitutions in FLT3 known to confer resistance to TKIs from clinical trials shows that substituting arginine for glycine at position 697 likely leads to steric clashes with FLT3 TKIs. It has been proposed that the F691 residue forms a stabilizing π-π stacking interaction with AC220, and thus a mutation to a Leu accounts for reduced binding affinity for this TKI. TTT-3002 is predicted to bind FLT3 without making direct contact with the F691 residue, and thus is unaffected by mutations at this site accounting for its continued potency. The closer proximity of F691 and N676 to sorafenib compared to staurosporine likely underlies the trend for substitutions at these sites to confer resistance to sorafenib-like but not staurosporine-like inhibitors. In summary, TTT-3002 has the potential for increased clinical applicability due to its ability to target not only FLT3/ITD and FLT3/PM AML patients but also patients who have developed resistance to other FLT3 TKIs through the selection of resistance mutations within the FLT3/ITD allele. These preclinical studies further support the entry of TTT-3002 into human clinical trials. Disclosures: Levis: Ambit: Consultancy.

scholarly journals K-RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells

Blood ◽  
2006 ◽  
Vol 109 (9) ◽  
pp. 3945-3952 ◽  
Author(s):  
Margaret E. M. Van Meter ◽  
Ernesto Díaz-Flores ◽  
Joehleen A. Archard ◽  
Emmanuelle Passegué ◽  
Jonathan M. Irish ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Fundamental Problem ◽  
Point Mutations ◽  
Tumor Formation ◽  
Signaling Networks ◽  
Hematopoietic Stem ◽  
Targeted Interventions ◽  
Ras Genes

Abstract Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers, including myeloid malignancies. We investigated the effects of an oncogenic KrasG12D allele on phosphorylated signaling molecules in primary c-kit+ lin−/low hematopoietic stem/progenitor cells. Comparison of wild-type and KrasG12D c-kit+ lin−/low cells shows that K-RasG12D expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5, ERK, and S6 under basal and stimulated conditions. Whereas KrasG12D cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor, we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell–targeted interventions.

scholarly journals Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

2019 ◽  
Author(s):  
Marie R Mooney ◽  
Erica E Davis ◽  
Nicholas Katsanis
Keyword(s):  
De Novo ◽  
Point Mutations ◽  
Enzyme Engineering ◽  
Single Nucleotide Variants ◽  
Guide Rna ◽  
Whole Exome ◽  
Two Generations ◽  
Sgrna Design ◽  
Favorable Conditions

AbstractTherapeutic applications of CRISPR-Cas9 gene editing have spurred innovation in Cas9 enzyme engineering and single guide RNA (sgRNA) design algorithms to minimize potential off-target events. While recent work in rodents outlines favorable conditions for specific editing and uses a trio design to control for the contribution of natural genome variation, the potential for CRISPR-Cas9 to induce de novo mutations in vivo remains a topic of interest. In zebrafish, we performed whole exome sequencing (WES) on two generations of offspring derived from the same founding pair: 54 exomes from control and CRISPR-Cas9 edited embryos in the first generation (F0), and 16 exomes from the progeny of inbred F0 pairs in the second generation (F1). We did not observe an increase in the number of transmissible variants in edited individuals in F1, nor in F0 edited mosaic individuals, arguing that in vivo editing does not precipitate an inflation of deleterious point mutations.

scholarly journals MCM2-7 complex is a novel druggable target for neuroendocrine prostate cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
En-Chi Hsu ◽  
Michelle Shen ◽  
Merve Aslan ◽  
Shiqin Liu ◽  
Manoj Kumar ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
De Novo ◽  
Treatment Resistance ◽  
Dna Helicase ◽  
Tumor Xenograft ◽  
Primary Tumors ◽  
Neuroendocrine Prostate Cancer ◽  
And Migration

AbstractNeuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer that rarely develops de novo in primary tumors and is commonly acquired during the development of treatment resistance. NEPC is characterized by gain of neuroendocrine markers and loss of androgen receptor (AR), making it resistant to current therapeutic strategies targeting the AR signaling axis. Here, we report that MCM2, MCM3, MCM4, and MCM6 (MCM2/3/4/6) are elevated in human NEPC and high levels of MCM2/3/4/6 are associated with liver metastasis and poor survival in prostate cancer patients. MCM2/3/4/6 are four out of six proteins that form a core DNA helicase (MCM2-7) responsible for unwinding DNA forks during DNA replication. Inhibition of MCM2-7 by treatment with ciprofloxacin inhibits NEPC cell proliferation and migration in vitro, significantly delays NEPC tumor xenograft growth, and partially reverses the neuroendocrine phenotype in vivo. Our study reveals the clinical relevance of MCM2/3/4/6 proteins in NEPC and suggests that inhibition of MCM2-7 may represent a new therapeutic strategy for NEPC.

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