Differential Localization of RAC1 and RAC2 Reflects Their Specific Functions in Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells.
Authors: Capala Marta Ewa , Maat Henny , Bonardi Francesco , Vellenga Edo , Schuringa Jan Jacob 
Published: October 2019

Abstract
Abstract 2302
Hematopoietic stem cells (HSCs) depend on the bone marrow niche to provide signals for their survival, quiescence and differentiation. Many of these microenvironmental signals converge on RAC GTPases. In the hematopoietic system, two members of the RAC family are expressed, RAC1 and RAC2. Although RAC1 and RAC2 share a very high sequence homology, specific functions of these proteins have been suggested. However, little has been revealed about the downstream effectors and molecular mechanisms. In this study, we used multiple approaches to gain insight into the molecular biology of RAC1 and RAC2 in normal and leukemic human HSCs. Firstly, GFP-tagged constructs of RAC1 and RAC2 were used to study localization of these proteins in CD34+/CD38−/Lin− HSCs. Time-lapse confocal imaging of living cells plated on stroma revealed that RAC1 was strongly enriched in the plasma membrane. In contrast, RAC2 localized predominantly in the cytoplasm of both resting and dividing HSCs, whereby localization changed dramatically when cells progressed from S to the G2 phase of the cell cycle. This very distinct localization pattern implied different functions of RAC1 and RAC2. Therefore, we specifically downregulated RAC1 and/or RAC2 to study the effects of their depletion in normal and BCR-ABL-transduced leukemic HSCs. In normal HSCs, simultaneous downregulation of RAC1 and RAC2 resulted in a modest but significant decrease in proliferation and progenitor frequencies in the long term stromal co-cultures. However, in BCR-ABL-transduced HSCs depletion of RAC2 alone, but not RAC1, was sufficient to induce a marked proliferative disadvantage, decreased progenitor frequency, reduced leukemic cobblestone formation and diminished replating capacity. To elucidate the mechanisms involved in the observed phenotypes, we employed an in vivo biotin labeling strategy of Avi-tagged RAC1 and RAC2 followed by pull down and mass-spectrometry to identify specific interaction partners of RAC1 and RAC2 in BCR-ABL-expressing hematopoietic cells. Several of the RAC1-specific interaction partners were annotated as plasma membrane proteins, involved in cell adhesion, cytoskeleton assembly and regulation of endocytosis. In contrast, RAC2-interacting proteins were cytoplasmic and involved in processes such as cell cycle progression, mitosis and regulation of apoptosis. Consistently with these findings, confocal time-lapse imaging of living hematopoietic cells revealed that pharmacological inhibition of RAC2 activity resulted in greatly decreased frequency of cell division. Moreover, the average division time was significantly extended upon RAC2 inhibition. Further functional characterization of RAC1 and RAC2-specific interactions is currently ongoing and will be discussed, but our data clearly indicate that distinct subcellular localization of RAC1 and RAC2 dictates their interaction with specific sets of proteins and consequently their specific functions in hematopoietic cells.

Disclosures:
No relevant conflicts of interest to declare.