UC San Diego

RASSF2 is a powerful pro-apoptotic K-Ras effector that is that is inactivated in many tumors via promoter methylation and has been shown to function as a tumor suppressor in lung, colorectal, and breast cell lines. RASSF2 belongs to the Ras-association domain family (RASSF) of proteins, which are able to engage in homo/hetero-dimerization and interact with common binding partners. In the context of acute myelogenous leukemia (AML), RASSF2 is exclusively downregulated in t(8;21) AML, suggesting that its repression may be essential for t(8;21) leukemia development.

In order to further characterize RASSF2’s tumor suppressive role in a leukemic context, we performed a CRISPR/Cas9-mediated knockout of RASSF2 in two non-t(8;21) AML cell lines: HL-60 and U937, generating single cell isolated clonal lines that are RASSF2 wild type, heterozygous, and knockout. Among the clonal cell lines, we assayed for changes in proliferation, apoptosis, cell cycle, and differentiation. We observed that heterozygous knockdown of RASSF2 in the U937 cells resulted in significantly higher (p<.05) proliferation when compared to wild type U937 clonal cells. However, mono- and bi-allelic knockout of RASSF2 did not result in significant differences in differentiation, apoptosis, or cell cycle arrest. In fact, clonal lines of the same genotype were observed to be characteristically variable. Taken together, we demonstrate that RASSF2 knockout is not critical in acute myelogenous leukemia since it does not manifest in a significant functional phenotype that overcame clonal characteristic variation. This suggests that loss of RASSF2 expression leads to the rescue of its function. We also prove that the process of single cell clonal isolation selects for clonal lines that are inherently variable in differentiation, which demonstrates that functional characterizations involving single cell clonal isolation must be performed with a larger pool of samples to account for the characteristic variability of clonal cell lines.