Identification of NRAS Downstream Genes with CRISPR Activation Screening

Akiya Tatsumi; Haruka Hirakochi; Satomi Inoue; Yosuke Tanaka; Hidehiro Furuno; Masumi Ikeda; Sachiko Ishibashi; Towako Taguchi; Kouhei Yamamoto; Iichiroh Onishi; Zohar Sachs; David A. Largaespada; Masanobu Kitagawa; Morito Kurata

doi:10.3390/biology11111551

Identification of NRAS Downstream Genes with CRISPR Activation Screening

Akiya Tatsumi, Haruka Hirakochi, Satomi Inoue, Yosuke Tanaka, Hidehiro Furuno, Masumi Ikeda, Sachiko Ishibashi, Towako Taguchi, Kouhei Yamamoto, Iichiroh Onishi, Zohar Sachs, David A. Largaespada, Masanobu Kitagawa, Morito Kurata

Research output: Contribution to journal › Article › peer-review

Abstract

Mutations in NRAS constitutively activate cell proliferation signaling in malignant neoplasms, such as leukemia and melanoma, and the clarification of comprehensive downstream genes of NRAS might lead to the control of cell-proliferative signals of NRAS-driven cancers. We previously established that NRAS expression and proliferative activity can be controlled with doxycycline and named as THP-1 B11. Using a CRISPR activation library on THP-1 B11 cells with the NRAS-off state, survival clones were harvested, and 21 candidate genes were identified. By inducting each candidate guide RNA with the CRISPR activation system, DOHH, HIST1H2AC, KRT32, and TAF6 showed higher cell-proliferative activity. The expression of DOHH, HIST1H2AC, and TAF6 was definitely upregulated with NRAS expression. Furthermore, MEK inhibitors resulted in the decreased expression of DOHH, HIST1H2AC, and TAF6 proteins in parental THP-1 cells. The knockdown of DOHH, HIST1H2AC, and TAF6 was found to reduce proliferation in THP-1 cells, indicating that they are involved in the downstream proliferation of NRAS. These molecules are expected to be new therapeutic targets for NRAS-mutant leukemia cells.

Original language	English (US)
Article number	1551
Journal	Biology
Volume	11
Issue number	11
DOIs	https://doi.org/10.3390/biology11111551
State	Published - Nov 2022

Bibliographical note

Funding Information:
D.A. Largaespada reports grants from the American Cancer Society during the conduct of the study; grants from Genentech, Inc.; other support from Luminary Therapeutics, Inc., Recombinetics, Inc., Makana Therapeutics, Inc., ImmuSoft, Inc., Styx Biotechnologies, Inc., andNeoClone Biotechnologies International; and personal fees from Bio-Techne, Inc., outside the submitted work. No disclosures were reported by the other authors.

Funding Information:
This research was funded by a Grant-in-Aid for Scientific Research (C) (18K06953) from the Japan Society for the Promotion of Science and the Kawano Masanori Memorial Foundation for the Promotion of Pediatrics.

Publisher Copyright:
© 2022 by the authors.

Keywords

CRISPR screening
NRAS

PubMed: MeSH publication types

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title = "Identification of NRAS Downstream Genes with CRISPR Activation Screening",

abstract = "Mutations in NRAS constitutively activate cell proliferation signaling in malignant neoplasms, such as leukemia and melanoma, and the clarification of comprehensive downstream genes of NRAS might lead to the control of cell-proliferative signals of NRAS-driven cancers. We previously established that NRAS expression and proliferative activity can be controlled with doxycycline and named as THP-1 B11. Using a CRISPR activation library on THP-1 B11 cells with the NRAS-off state, survival clones were harvested, and 21 candidate genes were identified. By inducting each candidate guide RNA with the CRISPR activation system, DOHH, HIST1H2AC, KRT32, and TAF6 showed higher cell-proliferative activity. The expression of DOHH, HIST1H2AC, and TAF6 was definitely upregulated with NRAS expression. Furthermore, MEK inhibitors resulted in the decreased expression of DOHH, HIST1H2AC, and TAF6 proteins in parental THP-1 cells. The knockdown of DOHH, HIST1H2AC, and TAF6 was found to reduce proliferation in THP-1 cells, indicating that they are involved in the downstream proliferation of NRAS. These molecules are expected to be new therapeutic targets for NRAS-mutant leukemia cells.",

keywords = "CRISPR screening, NRAS",

author = "Akiya Tatsumi and Haruka Hirakochi and Satomi Inoue and Yosuke Tanaka and Hidehiro Furuno and Masumi Ikeda and Sachiko Ishibashi and Towako Taguchi and Kouhei Yamamoto and Iichiroh Onishi and Zohar Sachs and Largaespada, {David A.} and Masanobu Kitagawa and Morito Kurata",

note = "Funding Information: D.A. Largaespada reports grants from the American Cancer Society during the conduct of the study; grants from Genentech, Inc.; other support from Luminary Therapeutics, Inc., Recombinetics, Inc., Makana Therapeutics, Inc., ImmuSoft, Inc., Styx Biotechnologies, Inc., andNeoClone Biotechnologies International; and personal fees from Bio-Techne, Inc., outside the submitted work. No disclosures were reported by the other authors. Funding Information: This research was funded by a Grant-in-Aid for Scientific Research (C) (18K06953) from the Japan Society for the Promotion of Science and the Kawano Masanori Memorial Foundation for the Promotion of Pediatrics. Publisher Copyright: {\textcopyright} 2022 by the authors.",

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month = nov,

doi = "10.3390/biology11111551",

language = "English (US)",

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AU - Tatsumi, Akiya

AU - Hirakochi, Haruka

AU - Inoue, Satomi

AU - Tanaka, Yosuke

AU - Furuno, Hidehiro

AU - Ikeda, Masumi

AU - Ishibashi, Sachiko

AU - Taguchi, Towako

AU - Yamamoto, Kouhei

AU - Onishi, Iichiroh

AU - Sachs, Zohar

AU - Largaespada, David A.

AU - Kitagawa, Masanobu

AU - Kurata, Morito

N1 - Funding Information: D.A. Largaespada reports grants from the American Cancer Society during the conduct of the study; grants from Genentech, Inc.; other support from Luminary Therapeutics, Inc., Recombinetics, Inc., Makana Therapeutics, Inc., ImmuSoft, Inc., Styx Biotechnologies, Inc., andNeoClone Biotechnologies International; and personal fees from Bio-Techne, Inc., outside the submitted work. No disclosures were reported by the other authors. Funding Information: This research was funded by a Grant-in-Aid for Scientific Research (C) (18K06953) from the Japan Society for the Promotion of Science and the Kawano Masanori Memorial Foundation for the Promotion of Pediatrics. Publisher Copyright: © 2022 by the authors.

PY - 2022/11

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N2 - Mutations in NRAS constitutively activate cell proliferation signaling in malignant neoplasms, such as leukemia and melanoma, and the clarification of comprehensive downstream genes of NRAS might lead to the control of cell-proliferative signals of NRAS-driven cancers. We previously established that NRAS expression and proliferative activity can be controlled with doxycycline and named as THP-1 B11. Using a CRISPR activation library on THP-1 B11 cells with the NRAS-off state, survival clones were harvested, and 21 candidate genes were identified. By inducting each candidate guide RNA with the CRISPR activation system, DOHH, HIST1H2AC, KRT32, and TAF6 showed higher cell-proliferative activity. The expression of DOHH, HIST1H2AC, and TAF6 was definitely upregulated with NRAS expression. Furthermore, MEK inhibitors resulted in the decreased expression of DOHH, HIST1H2AC, and TAF6 proteins in parental THP-1 cells. The knockdown of DOHH, HIST1H2AC, and TAF6 was found to reduce proliferation in THP-1 cells, indicating that they are involved in the downstream proliferation of NRAS. These molecules are expected to be new therapeutic targets for NRAS-mutant leukemia cells.

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Identification of NRAS Downstream Genes with CRISPR Activation Screening

Abstract

Bibliographical note

Keywords

PubMed: MeSH publication types

UN SDGs

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