Mutations in RAS family are observed in 6-96% of cancer. In acute myeloid leukemia (AML), mutations in NRAS/KRAS are seen in 10-20% of patients. NRAS proteins are a GDP-GTP regulator and have an important role in proliferative signaling networks such as PI3K and MAPK. However, the role of the magnitude of RAS activation in proliferation and cell cycle progression is not fully understood. To define the role of oncogenic NRAS dose in malignant behavior, we used a genetically engineered THP-1 cell line where the expression of NRASG12V is under the control of a tetracycline-responsive element. In this cell line, doxycycline (Dox) treatment induces expression of NRASG12V in a dose-dependent manner. THP-1 is a NRASG12D-dependent human AML cell line. We used CRISPR/Cas9 to knock out the endogenous NRASG12D and introduced a tetracycline-inducible NRASG12V. We established two modified cell lines: B11 and G6. When grown in the absence of Dox, B11 and G6 are both slow-growing and display high levels of G1 arrest. Growth in 100-1,000 ng/ml of Dox induces NRASG12V expression at levels comparable to native NRASG12D expression in unmodified THP-1 (at the protein and RNA level). To decipher the contribution of NRASG12V dose to cell cycle status, we treated B11 and G6 cell lines with three doses of Dox (0, 1,000, and 10,000 ng/ml) and performed mass cytometry (CyTOF) analysis. Native levels of NRASG12V (1,000 ng/ml Dox) led to maximal cell cycle progression with minimal G1 arrest. Interestingly higher levels of NRASG12V (Dox 10,000 ng/ml) led to reduction in cell cycle progression and an increase in G1 arrest. These data suggest that optimal levels of NRASG12V are required to optimally mediate leukemic activity. Using mass cytometry to assess signaling pathway activation, we also found that optimal NRASG12V maximally activated STAT and p38 pathways, suggesting that these may be critical mediators of NRASG12V-mediated cell cycle progression and self-renewal. These data reveal a dose-dependent role of oncogenic NRAS in mediating the proliferation and demonstrate that excessive oncogenic signaling may inhibit leukemogenic activity. To identify the whole downstream of NRAS signals, CRISPR knockout library, and CRISPR activation library were introduced into B11 and removing Dox to collect clone that can proliferate without NRAS signaling. As a result, 21 NRAS signal-related candidate genes were obtained from the knockout library and 28 from the activation library. DOHH is one of the candidates for CRISPR activation library screening. NRAS activation with Dox can induce the mRNA expression for DOHH in B11. DOHH is newly identified as a downstream of NRAS signaling. Taken together, the doxycycline-NRAS-controllable system can be applied for multiple-application to investigate the whole RAS signaling system.

Citation Format: Morito Kurata, Marie L. Antony, Klara E. Noble, Susan K. Rathe, Haruka Hirakouchi, Kouhei Yamamoto, Masanobu Kitagawa, Zohar Sachs, David A. Largaespada. Dose-dependent NRASG12V-mediated signaling controls cell cycle progression and leukemogenic signaling in a CRISPR/Cas9-modified human AML cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2582.