RNAi Screening of the Tyrosine Kinome Identifies Therapeutic Targets in Leukemia Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 758-758
Author(s):  
Jeffrey W Tyner ◽  
Marc Loriaux ◽  
Stephanie G Willis ◽  
Bill Chang ◽  
Vincent T Bicocca ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Tyrosine Kinases ◽  
White Blood Cells ◽  
Functional Assay ◽  
Functional Screening ◽  
Whole Genome ◽  
Primary Cells ◽  
Gene Target

Abstract A large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of cancer, but identification of specific tyrosine kinases as cancer targets has been a slow process. In the near future, whole-genome sequencing will enable vast amounts of sequence data to be collected, however clinical application of this information will require a detailed understanding of the functional consequences of each sequence change. Here, we present an RNAi-assisted protein target identification (RAPID) assay by which cells from leukemia patients are functionally screened with siRNA to determine tyrosine kinases that constitute amenable targets for therapeutic intervention. These data have led to identification of novel oncogenic anomalies in cancer patients. Combination of the RAPID screen with whole-genome sequencing promises to yield a powerful synthesis of methodologies by which both functional targets and genetic lesions can be rapidly determined. Methods: To detect targets necessary for viability of malignant cells, we screened primary cells from 75 patients with AML, ALL, CMML, and other MPD as well as white blood cells from healthy individuals by electroporating siRNAs individually targeting each member of the tyrosine kinase family. Four days later, we determined the cell viability and tabulated sensitivity of the cells to any individual tyrosine kinase. Where possible, results were confirmed by treating samples with small-molecule inhibitors with activity against the genes identified by the assay. In addition, the mechanism of oncogenesis was investigated for each positive result. Results: We demonstrate that siRNA screening can identify known oncogenic lesions such as K-RasG13D and JAK2V617F in primary cells from leukemia patients. The RAPID screen has also directed us towards a novel insertional mutation in the thrombopoietin receptor, MPL (1886InsGG). Additionally, we have detected FLT3 sensitivity in patients with FLT3-ITD and loss of heterozygosity, although not in FLT3-ITD heterozygous patients. In total, of 75 patients screened, this assay has yielded 25 cases that exhibit sensitivity to one or more tyrosine kinases. The mechanism of oncogenesis and its relation to the gene target has been established in select other samples with genetic abnormalities including evidence of chromosomal rearrangements as well as gene overexpression and mis-spicing events. Conclusions: We demonstrate that RNAi functional screening can determine sensitivity to individual tyrosine kinases in primary samples. Thus, this technique offers the potential to match specific therapies for targeted intervention with individual patients based on a functional assay. Additionally, in many cases, combination of the RAPID screen with whole-genome sequencing will enable efficient discovery of the genetic etiology of cancer.

scholarly journals RNAi screening of the tyrosine kinome identifies therapeutic targets in acute myeloid leukemia

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2238-2245 ◽  
Author(s):  
Jeffrey W. Tyner ◽  
Denise K. Walters ◽  
Stephanie G. Willis ◽  
Mary Luttropp ◽  
Jason Oost ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Cancer Genetics ◽  
Janus Kinase ◽  
Functional Assay ◽  
Functional Screening ◽  
Screening Assay ◽  
Activating Mutations ◽  
Rnai Technology ◽  
Tyrosine Kinome

Despite vast improvements in our understanding of cancer genetics, a large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of numerous types of cancer, but identification and validation of tyrosine kinase targets in cancer can be a time-consuming process. We report the establishment of an efficient, functional screening assay using RNAi technology to directly assess and compare the effect of individually targeting each member of the tyrosine kinase family. We demonstrate that siRNA screening can identify tyrosine kinase targets containing activating mutations in Janus kinase (JAK) 3 (A572V) in CMK cells and c-KIT (V560G) in HMC1.1 cells. In addition, this assay identifies targets that do not contain mutations, such as JAK1 and the focal adhesion kinases (FAK), that are crucial to the survival of the cancer cells. This technique, with additional development, might eventually offer the potential to match specific therapies with individual patients based on a functional assay.

RAPID siRNA Screen for Identification of Therapeutic Gene Targets in Patients with Hematologic Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3978-3978
Author(s):  
Jeffrey W Tyner ◽  
Marc Loriaux ◽  
Stephanie G Willis ◽  
Bill H Chang ◽  
Vincent T Bicocca ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Small Molecule ◽  
Therapeutic Intervention ◽  
Tyrosine Kinases ◽  
Research Funding ◽  
Hematologic Malignancies ◽  
Patient Specific ◽  
Functional Screening ◽  
Primary Cells ◽  
Gene Target

Abstract Abstract 3978 Poster Board III-914 A large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of cancer, but identification of specific kinases as cancer targets has been a slow process. Inhibition of cancer-causing tyrosine kinases offers a promising avenue of therapy, however this strategy of targeted therapy will require a detailed understanding of the oncogenic targets in each cancer patient. Here, we present an RNAi-assisted protein target identification (RAPID) assay by which cells from leukemia patients are functionally screened with siRNA to determine tyrosine kinases that constitute amenable targets for therapeutic intervention. Combination of the RAPID screen with gene-specific therapeutic approaches promises to yield a powerful synthesis of methodologies by which cancer patients can be specifically treated on the basis of functionally diagnosed gene targets. Methods To detect gene targets necessary for viability of malignant cells, we screened primary cells from 150 patients with hematologic malignancies by electroporating siRNAs individually targeting each member of the tyrosine kinase gene family. Four days later, we measured cell viability and tabulated sensitivity to silencing of specific genes. Samples were also screened for sensitivity to small-molecule kinase inhibitors. The mechanism of oncogenesis was investigated for each positive result. Results In total, we have identified 40 patient-specific gene targets in primary leukemia samples. We demonstrate that siRNA screening can identify known oncogenic lesions such as K-RasG13D and JAK2V617F in primary cells from leukemia patients. The RAPID screen has also directed us towards a novel insertional mutation in the thrombopoietin receptor, MPL (1886InsGG). Additionally, we have detected FLT3 sensitivity in patients with FLT3-ITD and loss of heterozygosity, although not in FLT3-ITD heterozygous patients. Agreement between siRNA-sensitive gene targets and small-molecule inhibitor sensitivity profiles has been high. The mechanism of oncogenesis and its relation to the gene target has been established in select other samples with abnormalities including gene overexpression and patient-specific mis-splicing events. Conclusions We demonstrate that RNAi functional screening can determine sensitivity to individual genes in cells obtained directly from cancer patients. Thus, this technique offers the potential to match targeted therapies with patients in a personalized manner. Application of these technologies will enable efficient discovery of the genetic etiology of cancer as well as a means for gene-specific therapeutic intervention. Disclosures: Deininger: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Calistoga: Research Funding; Genzyme: Research Funding. Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding.

scholarly journals Reliability of Spike Gene Target Failure for ascertaining SARS-CoV-2 lineage B.1.1.7 prevalence in a hospital setting

2021 ◽  
Author(s):  
José Afonso Guerra-Assunção ◽  
Paul A. Randell ◽  
Florencia A. T. Boshier ◽  
Michael A. Crone ◽  
Juanita Pang ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Lower Cost ◽  
Hospital Setting ◽  
Qpcr Assay ◽  
Spike Protein ◽  
Whole Genome ◽  
Gene Target ◽  
Spike Gene ◽  
The Uk

AbstractThe appearance of the SARS-CoV-2 lineage B.1.1.7 in the UK in late 2020, associated with faster transmission, sparked the need to find effective ways to monitor its spread. The set of mutations that characterise this lineage include a deletion in position 69 and 70 of the spike protein, which is known to be associated with Spike Gene Target Failure (SGTF) in a commonly used three gene diagnostic qPCR assay. The lower cost and faster turnaround times compared to whole genome sequencing make the use of qPCR for monitoring of the variant spread an attractive proposition. However, there are several potential issues with this approach. Here we use 826 SARS-CoV-2 samples collected in a hospital setting as part of the Hospital Onset COVID Infection (HOCI) study where qPCR was used for viral detection, followed by whole genome sequencing (WGS), to identify the factors to consider when using SGTF to infer lineage B.1.1.7 prevalence in a hospital setting, with potential implications for locations where this variant has recently been introduced.

scholarly journals Whole Genome Sequencing of Familial Non-Medullary Thyroid Cancer Identifies Germline Alterations in MAPK/ERK and PI3K/AKT Signaling Pathways

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 605 ◽  
Author(s):  
Aayushi Srivastava ◽  
Abhishek Kumar ◽  
Sara Giangiobbe ◽  
Elena Bonora ◽  
Kari Hemminki ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Whole Genome Sequencing ◽  
Signaling Pathways ◽  
Genome Sequencing ◽  
Tyrosine Kinases ◽  
Medullary Thyroid Cancer ◽  
Familial Cancer ◽  
Whole Genome ◽  
Medullary Thyroid ◽  
Genetic Burden

Evidence of familial inheritance in non-medullary thyroid cancer (NMTC) has accumulated over the last few decades. However, known variants account for a very small percentage of the genetic burden. Here, we focused on the identification of common pathways and networks enriched in NMTC families to better understand its pathogenesis with the final aim of identifying one novel high/moderate-penetrance germline predisposition variant segregating with the disease in each studied family. We performed whole genome sequencing on 23 affected and 3 unaffected family members from five NMTC-prone families and prioritized the identified variants using our Familial Cancer Variant Prioritization Pipeline (FCVPPv2). In total, 31 coding variants and 39 variants located in upstream, downstream, 5′ or 3′ untranslated regions passed FCVPPv2 filtering. Altogether, 210 genes affected by variants that passed the first three steps of the FCVPPv2 were analyzed using Ingenuity Pathway Analysis software. These genes were enriched in tumorigenic signaling pathways mediated by receptor tyrosine kinases and G-protein coupled receptors, implicating a central role of PI3K/AKT and MAPK/ERK signaling in familial NMTC. Our approach can facilitate the identification and functional validation of causal variants in each family as well as the screening and genetic counseling of other individuals at risk of developing NMTC.

scholarly journals Whole-genome sequencing of SARS-COV-2 reveals a substantially lower frequency of the UK variant than previously announced in Libya

2021 ◽  
Author(s):  
Inas M Alhudiri ◽  
Ahmad M Ramadan ◽  
Khaled Ibrahim Ibrahim ◽  
Mouna Eljilani ◽  
Adel Abdalla Aboud ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Drop Out ◽  
Whole Genome ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Gene Target ◽  
Specific Identification ◽  
S Gene ◽  
The Uk

A cluster-5 variant was detected in September 2020 in minks and humans in Denmark and currently classified as Alpha or B.1.1.7 strain. This variant presents several mutations in the spike region (S) which could increase the transmissibility of the virus 43-90% over previously circulating variants. The national center for disease control (NCDC) announced on 24th February 2021 the discovery of B.1.1.7 strain in Libya using a reverse-transcriptase PCR assay for S-gene target failure (SGTF) and reported that 25% of the tested samples were UK variant. This assay relies on the specific identification of the H69-V70 deletion in S gene which causes S gene drop out in RT-PCR; characteristic of the UK variant (B.1.1.7). This letter discusses our whole genome sequencing results of positive SARS-COV-2 samples with SGTF collected between 25th February - 4th March 2021 in Libya.

scholarly journals S Gene Target Failure (SGTF) in Commercial Multiplex RT-PCR assay as indicator to detect SARS-CoV-2 VOC B.1.1.7 lineage in Tamil Nadu, India

2021 ◽  
Author(s):  
Vidhya N M ◽  
Kumaresan A ◽  
Kalaivani V ◽  
Rajesh Kumar A ◽  
Gurunathan Subramanian ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Tamil Nadu ◽  
Immune Escape ◽  
Whole Genome ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Gene Target ◽  
S Gene ◽  
Significant Public Health

Emergence of Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) Variants of Concern (VOC) possessing improved virulence, transmissibility and/or immune-escape capabilities has raised significant public health concerns. In order to identify VOCs, WHO recommends Whole-Genome Sequencing approach, which is costly and involves longer completion time. Hence, potential role of commercial multiplex RT-PCR kit to screen variants rapidly is being attempted in this study. A total of 1200 suspected COVID samples from different districts of Tamil Nadu State (India) were screened with Thermo TaqPath RT-PCR kit and Altona Realstar RT-PCR Assay kit. Among 1200 screened, S-gene target failure (SGTF) phenomenon were identified in 112 samples while testing with TaqPath RT-PCR Kit. 100% concordant results were observed between SGTF phenomenon and whole-genome sequencing (WGS) results in detecting SARS-CoV-2 VOC B.1.1.7. TaqPath RT-PCR assay testing can be utilized by laboratories to screen rapidly the VOC B.1.1.7 variants, thus enabling early detection of B.1.1.7 variant infection and transmission in population. This in turn will pave way to implement suitable preventive measures by appropriate authorities to control the transmission of the viral variant.

scholarly journals Detection and whole-genome sequencing of SARS-CoV-2 B.1.617.2 and B.1.351 variants of concern from Pakistan during the COVID-19 third wave

2021 ◽  
Author(s):  
Massab Umair ◽  
Aamer Ikram ◽  
Muhammad Salman ◽  
Nazish Badar ◽  
Syed Adnan Haider ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Large Scale ◽  
Local Population ◽  
Genomic Diversity ◽  
Whole Genome ◽  
Third Wave ◽  
Close Monitoring ◽  
Gene Target ◽  
Azad Kashmir

The viral lineages reflecting variants of concern have emerged worldwide and among them B.1.1.7 (Alpha), B.1351 (Beta) and B.1.617.2 (Delta) variants are the most significant ones and merit close monitoring. In Pakistan, very limited information is available on the circulation of these variants and only the alpha variant has been reported as the main circulating lineage. The objective of this study was to detect and explore the genomic diversity of B.1.351 and B.1.617.2 during the third wave in the indigenous population. During the current study, a total of 2274 samples were tested on real-time PCR for the presence of SARS-CoV-2 from May 14 to May 31, 2021, and among these, 17% were spike positive, whereas 83% of samples had the spike gene target failure (SGTF). From these spike positive samples, 22 samples were processed for whole-genome sequencing. Among VOCs, 45.5% (n=10) belonged to B.1.351 followed by B.1.617.2, 36% (n=8). The delta variant cases were reported mostly from Islamabad (n = 5; 63%) followed by Peshawar and Azad Kashmir (n = 1; 13% each). Beta variant cases originated from Islamabad (n=5; 56%), Peshawar (n=2; 22%), Azad Kashmir and Rawalpindi (n=1; 11% each). The mutation profile of delta variant isolates reported significant mutations, L452R, T478K, P681R, and D950N. The beta variant isolates reported characteristic mutations, D215G, K417N, E484K, N501Y, and A701V. Notably, a characteristic mutation, E484Q was also found in delta variant, B.1.617.2. Our current findings suggest detection of these VOCs from the local population and warrants large-scale genomic surveillance in the country. In addition, it provides timely information to the health authorities to take appropriate actions.

scholarly journals Whole Genome Sequencing of Familial Non-Medullary Thyroid Cancer Identifies Germline Alterations in MAPK/ERK and PI3K/AKT Signaling Pathways

2019 ◽  
Author(s):  
Aayushi Srivastava ◽  
Abhishek Kumar ◽  
Sara Giangiobbe ◽  
Elena Bonora ◽  
Kari Hemminki ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Whole Genome Sequencing ◽  
Signaling Pathways ◽  
Genome Sequencing ◽  
Tyrosine Kinases ◽  
Medullary Thyroid Cancer ◽  
Familial Cancer ◽  
Whole Genome ◽  
Medullary Thyroid ◽  
Genetic Burden

Evidence of familial inheritance in non-medullary thyroid cancer (NMTC) has accumulated over the last few decades. However, known variants account for a very small percentage of the genetic burden. Here, we focused on the identification of common pathways and networks enriched in NMTC families to better understand its pathogenesis with the final aim of identifying one novel high/moderate-penetrance germline predisposition variant segregating with the disease in each studied family. We performed whole genome sequencing on 23 affected and 3 unaffected family members from five NMTC-prone families and prioritized the identified variants using our Familial Cancer Variant Prioritization Pipeline (FCVPPv2). In total, 31 coding variants and 39 variants located in upstream, downstream, 5′ or 3′ untranslated regions passed FCVPPv2 filtering. Altogether, 210 genes affected by variants that passed the first three steps of the FCVPPv2 were analyzed using Ingenuity Pathway Analysis software. These genes were enriched in tumorigenic signaling pathways mediated by receptor tyrosine kinases and G-protein coupled receptors, implicating a central role of PI3K/AKT and MAPK/ERK signaling in familial NMTC. Our approach can facilitate the identification and functional validation of causal variants in each family as well as the screening and genetic counseling of other individuals at risk of developing NMTC.

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