High-throughput sequencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients

Abstract Transforming mutations in NRAS and KRAS are thought to play a causative role in the development of numerous cancers, including myeloid malignancies. Although mutations at amino acids 12, 13, or 61 account for the majority of oncogenic Ras variants, we hypothesized that less frequent mutations at alternate residues may account for disease in some patients with cancer of unexplained genetic etiology. To search for additional, novel RAS mutations, we sequenced all coding exons in NRAS, KRAS, and HRAS in 329 acute myeloid leukemia (AML) patients, 32 chronic myelomonocytic leukemia (CMML) patients, and 96 healthy individuals. We detected 4 “noncanonical” point mutations in 7 patients: N-RasG60E, K-RasV14I, K-RasT74P, and K-RasA146T. All 4 Ras mutants exhibited oncogenic properties in comparison with wild-type Ras in biochemical and functional assays. The presence of transforming RAS mutations outside of positions 12, 13, and 61 reveals that alternate mechanisms of transformation by RAS may be overlooked in screens designed to detect only the most common RAS mutations. Our results suggest that RAS mutations may play a greater role in leukemogenesis than currently believed and indicate that high-throughput screening for mutant RAS alleles in cancer should include analysis of the entire RAS coding region.

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High-throughput screening reveals novel mutations in spinal muscular atrophy patients

10.21203/rs.2.12903/v1 ◽

2019 ◽

Author(s):

Jianbo Shu ◽

Jingrui Wang ◽

Yulian Fang ◽

Zanmei Xu ◽

Xiaowei Wang ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

High Throughput ◽

High Throughput Screening ◽

High Throughput Sequencing ◽

Muscular Atrophy ◽

Point Mutations ◽

Control Group ◽

Compound Heterozygous ◽

Single Nucleotide Variants ◽

Illumina Hiseq

Abstract Background Some spinal muscular atrophy (SMA) cases are caused by either compound heterozygosity with a point mutation in one allele and a deletion in the other or compound heterozygous point mutations in SMN1 or other genes. Methods To explore more genes and mutations in the onset of SMA, 83 whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was firstly performed with a SALSA MLPA Kit P021 for preliminary diagnosis. Afterwards, the complete gene sequence of SMN1 gene was detected with the high-throughput sequencing platform of Illumina HiSeq-2500 to find more mutations in the 28 core families. Furthermore, 20 SMA patients were selected from the 28 prodands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results MLPA results showed that 22 probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. 6 SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C>T], c.[271C>T], c.[-39A>G] and g.[70240639G>C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[-41_-40insCTCT], FUT5 SNV c.[1001A>G], and MCCC2 SNV c.[-117A>G] were the 3 most frequent mutations in SMA group (95%, 85% and 75%, respectively). Conclusions We identified some mutations in both SMN1 and other genes, and c.[271C>T], c.[-41_-40insCTCT], c.[1001A>G] and c.[-117A>G] might be associated with the onset of SMA.

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High-throughput screening reveals novel mutations in spinal muscular atrophy patients

Italian Journal of Pediatrics ◽

10.1186/s13052-020-00925-1 ◽

2020 ◽

Vol 46 (1) ◽

Author(s):

Ruiping Zhang ◽

Chunyu Gu ◽

Linjie Pu ◽

Yingtao Meng ◽

Jianbo Shu ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

High Throughput ◽

High Throughput Screening ◽

High Throughput Sequencing ◽

Muscular Atrophy ◽

Hereditary Disease ◽

Control Group ◽

Single Nucleotide Variants ◽

Frequent Mutations ◽

Multiplex Ligation Probe Amplification

Abstract Background Spinal muscular atrophy (SMA) is an autosomal recessive hereditary disease associated with severe muscle atrophy and weakness in the limbs and trunk. The discovery of mutated genes is helpful in diagnosis and treatment for SMA. Methods Eighty-three whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was performed. Afterwards, the complete gene sequence of SMN1 gene was detected. Furthermore, 20 SMA patients were selected from the 28 probands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results Twenty-two probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. Six SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C > T], c.[271C > T], c.[−39A > G] and g.[70240639G > C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[−41_-40insCTCT], FUT5 SNV c.[1001A > G], and MCCC2 SNV c.[−117A > G] were the 3 most frequent mutations in SMA group (95, 85 and 75%, respectively). Conclusions We identified some mutations in both SMN1 and other genes, and c.[271C > T], c.[−41_-40insCTCT], c.[1001A > G] and c.[−117A > G] might be associated with the onset of SMA.

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High-throughput screening reveals novel mutations in spinal muscular atrophy patients

10.21203/rs.3.rs-21587/v1 ◽

2020 ◽

Author(s):

Ruiping Zhang ◽

Chunyu Gu ◽

Linjie Pu ◽

Yingtao Meng ◽

Jianbo Shu ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

High Throughput ◽

High Throughput Screening ◽

High Throughput Sequencing ◽

Muscular Atrophy ◽

Hereditary Disease ◽

Control Group ◽

Single Nucleotide Variants ◽

Frequent Mutations ◽

Multiplex Ligation Probe Amplification

Abstract Background Spinal muscular atrophy (SMA) is an autosomal recessive hereditary disease associated with severe muscle atrophy and weakness in the limbs and trunk. The discovery of genes mutated by SMA is helpful in diagnosis and treatment. Methods 83 whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was firstly performed. Afterwards, the complete gene sequence of SMN1 gene was detected. Furthermore, 20 SMA patients were selected from the 28 probands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results 22 probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. 6 SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C>T], c.[271C>T], c.[-39A>G] and g.[70240639G>C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[-41_-40insCTCT], FUT5 SNV c.[1001A>G], and MCCC2 SNV c.[-117A>G] were the 3 most frequent mutations in SMA group (95%, 85% and 75%, respectively). Conclusions We identified some mutations in both SMN1 and other genes, and c.[271C>T], c.[-41_-40insCTCT], c.[1001A>G] and c.[-117A>G] might be associated with the onset of SMA.

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High-throughput screening reveals novel mutations in spinal muscular atrophy patients

10.21203/rs.3.rs-21587/v2 ◽

2020 ◽

Author(s):

Ruiping Zhang ◽

Chunyu Gu ◽

Linjie Pu ◽

Yingtao Meng ◽

Jianbo Shu ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

High Throughput ◽

High Throughput Screening ◽

High Throughput Sequencing ◽

Muscular Atrophy ◽

Hereditary Disease ◽

Control Group ◽

Single Nucleotide Variants ◽

Frequent Mutations ◽

Multiplex Ligation Probe Amplification

Abstract Background: Spinal muscular atrophy (SMA) is an autosomal recessive hereditary disease associated with severe muscle atrophy and weakness in the limbs and trunk. The discovery of mutated genes is helpful in diagnosis and treatment for SMA. Methods: 83 whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was performed. Afterwards, the complete gene sequence of SMN1 gene was detected. Furthermore, 20 SMA patients were selected from the 28 probands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results: 22 probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. 6 SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C>T], c.[271C>T], c.[-39A>G] and g.[70240639G>C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[-41_-40insCTCT], FUT5 SNV c.[1001A>G], and MCCC2 SNV c.[-117A>G] were the 3 most frequent mutations in SMA group (95%, 85% and 75%, respectively). Conclusions: We identified some mutations in both SMN1 and other genes, and c.[271C>T], c.[-41_-40insCTCT], c.[1001A>G] and c.[-117A>G] might be associated with the onset of SMA.

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High-Throughput Screening for Induced Point Mutations

PLANT PHYSIOLOGY ◽

10.1104/pp.126.2.480 ◽

2001 ◽

Vol 126 (2) ◽

pp. 480-484 ◽

Cited By ~ 308

Author(s):

Trenton Colbert ◽

Bradley J. Till ◽

Rachel Tompa ◽

Steve Reynolds ◽

Michael N. Steine ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Point Mutations

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High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes

Life ◽

10.3390/life10050058 ◽

2020 ◽

Vol 10 (5) ◽

pp. 58

Author(s):

Aida Martinez-Sanchez ◽

Stefano Lazzarano ◽

Eshita Sharma ◽

Helen Lockstone ◽

Christopher L. Murphy

Keyword(s):

Cartilage Repair ◽

High Throughput ◽

High Throughput Sequencing ◽

Articular Chondrocytes ◽

Articular Chondrocyte ◽

Coding Region ◽

Cartilage Development ◽

Rna Immunoprecipitation ◽

Important Regulator ◽

Mmp13 Expression

MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3′UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

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FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia

Blood ◽

10.1182/blood.v97.11.3589 ◽

2001 ◽

Vol 97 (11) ◽

pp. 3589-3595 ◽

Cited By ~ 298

Author(s):

Derek L. Stirewalt ◽

Kenneth J. Kopecky ◽

Soheil Meshinchi ◽

Frederick R. Appelbaum ◽

Marilyn L. Slovak ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Clinical Outcomes ◽

Older Patients ◽

Myeloid Leukemia ◽

Point Mutations ◽

Tp53 Mutations ◽

Ras Mutations ◽

Cell Counts ◽

Single Stranded Conformational Polymorphism ◽

Acute Myeloid

The prevalence and significance of genetic abnormalities in older patients with acute myeloid leukemia (AML) are unknown. Polymerase chain reactions and single-stranded conformational polymorphism analyses were used to examine 140 elderly AML patients enrolled in the Southwest Oncology Group study 9031 for FLT3, RAS, and TP53 mutations, which were found in 34%, 19%, and 9% of patients, respectively. All but one of the FLT3 (46 of 47) mutations were internal tandem duplications (ITDs) within exons 11 and 12. In the remaining case, a novel internal tandem triplication was found in exon 11. FLT3 ITDs were associated with higher white blood cell counts, higher peripheral blast percentages, normal cytogenetics, and less disease resistance. All RAS mutations (28 of 28) were missense point mutations in codons 12, 13, or 61. RASmutations were associated with lower peripheral blast and bone marrow blast percentages. Only 2 of 47 patients with FLT3 ITDs also had a RAS mutation, indicating a significant negative association between FLT3 and RAS mutations (P = .0013). Most TP53 mutations (11 of 12) were missense point mutations in exons 5 to 8 and were associated with abnormal cytogenetics, especially abnormalities in both chromosomes 5 and 7. FLT3 and RAS mutations were not associated with inferior clinical outcomes, but TP53mutations were associated with a worse overall survival (median 1 versus 8 months, P = .0007). These results indicate that mutations in FLT3, RAS, or TP53 are common in older patients with AML and are associated with specific AML phenotypes as defined by laboratory values, cytogenetics, and clinical outcomes.

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HO-1 promotes resistance to an EZH2 inhibitor through the pRB-E2F pathway: correlation with the progression of myelodysplastic syndrome into acute myeloid leukemia

Journal of Translational Medicine ◽

10.1186/s12967-019-2115-9 ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 1

Author(s):

Zhengchang He ◽

Siyu Zhang ◽

Dan Ma ◽

Qin Fang ◽

Liping Yang ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

High Risk ◽

Myelodysplastic Syndrome ◽

Tumor Cells ◽

High Throughput ◽

Myeloid Leukemia ◽

High Throughput Sequencing ◽

Ezh2 Expression ◽

Very High ◽

Acute Myeloid

Abstract Background Myelodysplastic syndrome (MDS) can progress to acute myeloid leukemia (AML), and conventional chemotherapy (decitabine) does not effectively inhibit tumor cells. Enhancer of zeste homologue 2 (EZH2) and Heme oxygenase-1 (HO-1) are two key factors in patients resistance and deterioration. Methods In total, 58 MDS patients were divided into four groups. We analyzed the difference in HO-1 and EZH2 expression among the groups by real-time PCR. After treatment with Hemin or Znpp IX, flow cytometry was used to detect apoptosis and assess the cell cycle distribution of tumor cells. Following injection of mice with very high-risk MDS cells, spleen and bone marrow samples were studied by immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining. MDS cells overexpressing EZH2 and HO-1 were analyzed by high-throughput sequencing. The effect of HO-1 on the pRB-E2F pathway was analyzed by Western blotting. The effects of decitabine on P15INK4B and TP53 in MDS cells after inhibiting HO-1 were detected by Western blotting. Results Real-time PCR results showed that EZH2 and HO-1 expression levels were higher in MDS patients than in normal donors. The levels of HO-1 and EZH2 were simultaneously increased in the high-risk and very high-risk groups. Linear correlation analysis and laser scanning confocal microscopy results indicated that EZH2 was related to HO-1. MDS cells that highly expressed EZH2 and HO-1 infiltrated the tissues of experimental mice. IHC results indicated that these phenomena were related to the pRB-E2F pathway. High-throughput sequencing indicated that the progression of MDS to AML was related to EZH2. Using the E2F inhibitor HLM006474 and the EZH2 inhibitor JQEZ5, we showed that HO-1 could regulate EZH2 expression. HO-1 could stimulate the transcription and activation of EZH2 through the pRB-E2F pathway in MDS patients during chemotherapy, which reduced TP53 and P15INK4B expression. Conclusions EZH2 was associated with HO-1 in high-risk and very high-risk MDS patients. HO-1 could influence MDS resistance and progression to AML.

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Oncogenic RAS Enables DNA Damage- and p53-Dependent Differentiation of Acute Myeloid Leukemia Cells in Response to Chemotherapy.

Blood ◽

10.1182/blood.v114.22.5040.5040 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5040-5040

Author(s):

Mona Meyer ◽

Daniela Rübsamen ◽

Robert K. Slany ◽

Thomas Illmer ◽

Kathleen Stabla ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Chemotherapeutic Agents ◽

Myeloid Differentiation ◽

High Dose ◽

Ras Mutations ◽

Oncogenic Ras ◽

Response To Chemotherapy ◽

High Dose Cytarabine ◽

Acute Myeloid

Abstract Abstract 5040 Introduction Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and patients with RAS mutations benefit most from high-dose cytarabine as postremission therapy (Neubauer et al., J Clin Oncol 2008). The molecular reason for this phenomenon is not well understood. Methods We used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. In addition, we used primary human inversion 16 positive AML samples with or without oncogenic RAS mutations to corroborate our findings. Results There was no difference in cell cycle kinetics, apoptosis or cellular senescence in MLL-ENL cell expressing control vector vs. MLL-ENL cells expressing oncogenic RAS in response to cytarabine treatment. However, we observed an increased activation of DNA damage checkpoints in MLL-ENL-cells expressing oncogenic RAS after incubation with chemotherapeutic agents. This resulted in an Atm/r as well as p53-dependent genetic program causing dramatically reduction of clonogenicity in MLL-ENL cells expressing oncogenic RAS due to induction of myeloid differentiation. Co-expression of dominant – negative p53 completely abolished this differentiation resulting in the rescue of the clonogic potential. Activation of p53 as a result of inhibition of Mdm2-mediated degradation of p53 further enhances this myeloid differentiation. Of note, in primary AML cases, oncogenic RAS also was associated with a more differentiated phenotype. Conclusions The data can explain the beneficial effects observed in AML patients with oncogenic RAS mutations treated with high dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy since stronger acitvation of p53 by oncogenic RAS and chemotherapeutic agents cause the differentiation of hematopoietic stem cells to more differentiated progenitors. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells. Disclosures No relevant conflicts of interest to declare.

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Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

10.1101/208439 ◽

2017 ◽

Cited By ~ 1

Author(s):

Nathan P. Coussens ◽

Stephen C. Kales ◽

Mark J. Henderson ◽

Olivia W. Lee ◽

Kurumi Y. Horiuchi ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Point Mutations ◽

Set Domain ◽

Histone Lysine ◽

Histone Lysine Methyltransferase ◽

Attractive Therapeutic Target ◽

Lysine Methyltransferase ◽

In Cells

AbstractThe activity of the histone lysine methyltransferase NSD2 is thought to play a driving role in oncogenesis. Both overexpression of NSD2 and point mutations that increase its catalytic activity are associated with a variety of human cancers. While NSD2 is an attractive therapeutic target, no potent, selective and cell-active inhibitors have been reported to date, possibly due to the challenging nature of developing high-throughput assays for NSD2. To establish a platform for the discovery and development of selective NSD2 inhibitors, multiple assays were optimized and implemented. Quantitative high-throughput screening was performed with full-length wild-type NSD2 and a nucleosome substrate against a diverse collection of known bioactives comprising 16,251 compounds. Actives from the primary screen were further interrogated with orthogonal and counter assays, as well as activity assays with the clinically relevant NSD2 mutants E1099K and T1150A. Five confirmed inhibitors were selected for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. The identification of NSD2 inhibitors that bind the catalytic SET domain and demonstrate activity in cells validates the workflow, providing a template for identifying selective NSD2 inhibitors.

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