OCI-Aml-3

Proteomic Analysis of Nucleostemin in OCI-AML-3 Cell Line

Nucleostemin (NS; a product of the GNL3 gene) is a nucleolar-nucleoplasm shuttling GTPase whose levels are high in several solid and hematological neoplasms, including acute myeloid leukemia (AML). It plays a role in telomere maintenance, response to stress stimuli, and favoring DNA repair.

To investigate the role of NS in AML with NPM1 mutation, the OCI-AML 3 cell line bearing the most common NPM1 mutation was used as a model system. Preliminarily, basal NS expression in a panel of AML cell lines was assessed. First of all, the study observed a strong expression of NS in OCI-AML 3 SCR control cells, which was markedly reduced after 72 h of doxycycline treatment. NS protein levels were less than 10% in shNS OCI-AML 3 cells in comparison with the SCR control (Fig. 1A). In addition, mRNA expression levels of NS in silenced cells were significantly reduced by at least of 50%-as compared to the SCR control (Fig. 1B).

To further shed light on the role of NS expression in OCI-AML 3 cells, the study performed a shotgun proteomics analysis of shNS cells and their SCR control. 1298 proteins for OCI-AML 3 SCR and 1452 proteins for OCI-AML 3 shNS were identified and quantified. Among them, 1245 proteins were in common between the two conditions, as reported in the Venn diagram of Fig. 1C, consisting of 82.5% of all identified proteins. OCI-AML 3 cells with silenced NS significantly triggered biological processes related to down-regulation of cell movement, cell viability, and homologous recombination as well as significantly up-regulated mechanisms linked to cell death of cancer cells and bone marrow lesions.

Fig. 1 NS expression, silencing, and differential proteomics analysis of OCI-AML 3 cells. (Cela I, et al., 2022)

Pim Kinase Inhibitor Inducing Acute Myeloid Leukemia Cell Death

Pim kinases are serine/threonine kinases with multiple substrates that affect survival pathways. The imidazo pyridazine compound SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. It was evaluated in AML cell lines (MV-4-11, MOLM-13, and OCI-AML-3). The phosphorylation of traditional Pim kinase targets, c-Myc (Ser62), and 4E-BP1 (Thr36/Thr47) decreased in actively cycling AML cell lines MV-4-11, MOLM-13, and OCI-AML-3 (Fig. 2).

Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis. A significant reduction of levels of antiapoptotic proteins Mcl-1 was observed. In OCI-AML-3, RNA synthesis was inhibited to 80% and 50% of control at 1 and 10 μM SGI-1776, respectively (Fig. 3A). The MV-4-11 and MOLM-13 cell lines also showed inhibition of RNA synthesis, and further studies found that inhibition of RNA synthesis was associated with reduced levels of the MCL-1 transcript and impaired protein translation. MV-4-11, MOLM-13, and OCI-AML-3 cells were treated with SGI-1776 and then stained with either annexin/PI or DiOC6/PI to assess cell survival. There was a dose-dependent increase in annexin/PI-positive cells in all 3 lines (Fig. 4). To sum up, Pim kinase inhibition may be a new strategy for AML treatment.

Fig. 2 Immunoblot analysis of Pim kinase targets in AML primary cells treated with SGI-1776. (Chen LS, et al., 2011)

Fig. 3 Effect of SGI-1776 in AML cell lines. (Chen LS, et al., 2011)

Fig. 4 Induction of cell death by SGI-1776 in AML cell lines. (Chen LS, et al., 2011)