Identification of NRAS Downstream Genes with CRISPR Activation Screening

SIMPLE SUMMARY: NRAS mutations constitutively activate cell proliferation signaling in malignant tumors. The elucidation of NRAS downstream signaling genes may lead to the control of NRAS-mutant tumors. Genome-wide CRISPR activation screening was performed on THP-1 B11, in which NRAS expression and cell proliferation could be regulated by doxycycline. Multiple candidate genes were identified from clones that survived in NRAS-off, and three genes—DOHH, HIST1H2AC, and TAF6—were finally identified as being downstream of NRAS signaling and confirmed to contribute to the proliferation of THP-1 cells. These molecules are promising new therapeutic targets for NRAS-mutant leukemia. 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.