B11 cells created by replacing the NRAS-G12D mutation in THP1 cells with a dox-inducible NRAS-G12V mutation
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA476487 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7352470.fastq.gz | Fastqsanger.gz | |||
| SRR7352461.fastq.gz | Fastqsanger.gz | |||
| SRR7352471.fastq.gz | Fastqsanger.gz | |||
| SRR7352462.fastq.gz | Fastqsanger.gz | |||
| SRR7352472.fastq.gz | Fastqsanger.gz |
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Project description:To study the role of NRAS mutations in cell proliferation and self-renewal in acute myeloid leukemia (AML), the human AML cell line, THP1, was modified to replace its naturally occurring heterozygous NRAS-G12D mutation with a doxycycline(dox)-inducible heterozygous NRAS-G12V mutation. The endogenous copies of the NRAS-G12D allele were deleted using CRISPR/Cas9 after a dox-inducible, CRISPR resistant, NRAS-G12V transgene was introduced into the THP1 cell line. The resulting cell line was named B11. RNA-seq data confirmed that endogenous NRAS G12D was successfully replaced by dox-inducible exogenous NRAS G12V in the B11 cell line. As expected, depletion of dox induced G1 cell cycle arrest. Interestingly, the B11 cells experienced ten-times higher expression of NRAS induced G2/S-phase cell cycle arrest. Forty-nine genes were identified as signaling responsible genes associated with high expression of NRAS.
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Project description:The goal of this study was to determine genes affected by expressing KRAS mutation (G12V) in NCI-H1703 cells This data was used in Meng Wang et. al. Cancer Research 2016 to determine the alterations of gene expression profiling associated with expression of KRAS mutation (G12V).
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Project description:The design of potent RAS inhibitors benefits from a molecular understanding of the dynamics in KRAS and NRAS and their oncogenic mutants. Here we characterize switch-1 dynamics in GTP-state KRAS and NRAS by 31P NMR, by 15N relaxation dispersion NMR, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics simulations. In GMPPNP-bound KRAS and NRAS, we see the co-existence of two conformational states, corresponding to an “inactive” state-1 and an “active” state-2, as previously reported. The KRAS oncogenic mutations G12D, G12C and G12V only slightly affect this equilibrium towards the “inactive” state-1, with rank order wt < G12C < G12D < G12V. In contrast, the NRAS Q61R oncogenic mutation shifts the equilibrium fully towards the “active” state-2. Our molecular dynamics simulations explain this by the observation of a transient hydrogen bond between the Arg61 side chain and the Thr35 backbone carbonyl oxygen. NMR relaxation dispersion experiments with GTP-bound KRAS Q61R confirm a drastic decrease in the population of state-1, but still detect a small residual population (1.8%) of this conformer. HDX-MS indicates that higher populations of state-1 correspond to increased hydrogen-deuterium exchange rates in some regions and increased flexibility, whereas low state-1 populations are associated with KRAS rigidification. We elucidated the mechanism of action of a potent KRAS G12D inhibitor, MRTX1133. Binding of this inhibitor to the switch-2 pocket causes a complete shift of KRAS G12D towards the “inactive” conformation and prevents binding of effector RAS-binding domain (RBD), by signaling through an allosteric network.
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Project description:The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4-, CD8- ab T cells. Mutations in ALPS typically affect CD95 (Fas/APO-1)-mediated apoptosis, one of the extrinsic death pathways involving tumor necrosis factor receptor (TNFR) superfamily proteins, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair CD95-mediated apoptosis. The increase in active, GTP-bound NRAS augments RAF/MEK/ERK signaling which markedly decreases the pro-apoptotic protein BIM and attenuates intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects. Our observations on the effects of NRAS activation indicate that RAS-inactivating drugs, such as farnesyl-transferase inhibitors (FTIs) should be examined in human autoimmune and lymphocyte homeostasis disorders. Keywords: NRAS
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Project description:Despite KRAS-G12V being the second most common KRAS mutation in cancer, there are no approved direct KRAS-G12V inhibitors. Difficulties with inhibiting oncogenes using conventional approaches have prompted the use of RNAi as a therapeutic approach. RNAi has faced numerous obstacles as a cancer therapeutic, including the lack of cancer-specific tissue targeting, rapid oligonucleotide nuclease degradation and clearance from the circulation. Recently, the use of targetable ligands conjugated to chemically modified siRNAs has shown remarkable promise in circumventing these barriers. We demonstrate that EFTX-G12V is highly selective for KRAS-G12V and has improved therapeutic activity over pan-KRAS targeting, including enhanced inhibition of several cancer hallmarks. With a novel RNAi delivery platform, we demonstrate tumor silencing of KRAS-G12V and significant anti-tumor activity across several cancer models. Our findings represent a technologic advance in oncogene targeting using RNAi and reveal new biologic insights in KRAS targeting that may have broad implications with regards to safety and efficacy. This dataset collects the RNA-Seq data for the H441 cell line.
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Project description:Despite KRAS-G12V being the second most common KRAS mutation in cancer, there are no approved direct KRAS-G12V inhibitors. Difficulties with inhibiting oncogenes using conventional approaches have prompted the use of RNAi as a therapeutic approach. RNAi has faced numerous obstacles as a cancer therapeutic, including the lack of cancer-specific tissue targeting, rapid oligonucleotide nuclease degradation and clearance from the circulation. Recently, the use of targetable ligands conjugated to chemically modified siRNAs has shown remarkable promise in circumventing these barriers. We demonstrate that EFTX-G12V is highly selective for KRAS-G12V and has improved therapeutic activity over pan-KRAS targeting, including enhanced inhibition of several cancer hallmarks. With a novel RNAi delivery platform, we demonstrate tumor silencing of KRAS-G12V and significant anti-tumor activity across several cancer models. Our findings represent a technologic advance in oncogene targeting using RNAi and reveal new biologic insights in KRAS targeting that may have broad implications with regards to safety and efficacy. This dataset collects the RNA-Seq data for the SKCO1 cell line.
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