The Role of Ubiquitin E3 Ligase FBXO21 in Steady-State, Stressed, and Malignant Hematopoiesis

Hematopoietic stem cells (HSCs) allow for the formation of all cell types in the blood and maintain these populations throughout a person’s life. The process of hematopoiesis is regulated through a variety of molecular mechanisms that are either signaled from the cell’s environment (extrinsic) or from within the cell itself (intrinsic). One intrinsic mechanism that regulates hematopoietic cell fate decisions is the ubiquitin proteasome system (UPS). The UPS controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. The key component of the UPS is the ubiquitin E3 ligase as this protein is the substrate recognition component, the protein that decides which substrate will be chosen for degradation. Through investigating the FBOX family of ubiquitin E3 ligases, we identified FBXO21, the substrate recognition component of the SKP1-CUL1-FBOX (SCF) complex, as an important regulator of normal, stressed, and malignant hematopoiesis. We first identified FBXO21 to have high RNA expression and protein abundance in wild-type (WT) mouse hematopoietic stem and progenitor cells (HSPCs) compared with other hematopoietic populations. To study the role of FBXO21 in HSC maintenance and transformation, we utilized shRNAs designed to target FBXO21/Fbxo21 mRNA, and generated the first tissue specific Fbxo21 conditional knockout (cKO) mouse model. We found that loss of Fbxo21 in vivo led to minimal alterations in hematopoietic populations. However, a loss of Fbxo21 in vitro led to a decrease in certain HSPC cell populations and a decrease in HSCs ability to form colonies. Mass spectrometry (MS) analysis also revealed that loss of Fbxo21 led to an increase in proteins involved in the immune system process, the innate immune response, and leukocyte migration. Two of the proteins increased, S100A8&9 and CD177, are seen to act upstream of extracellular signal-regulated kinase (ERK) activation, which supports the increase in ERK activation we originally identified when Fbxo21 was knocked down. When we stressed the hematopoietic system of our Fbxo21 cKO mice through stimulation with granulocyte colony-stimulating factor (G-CSF), an injection of lipopolysaccharide (LPS), bone marrow (BM) transplantation, or serial injections of 5-fluorouracil (5-FU), we found that modulating Fbxo21 expression effected hematopoiesis only when mice were stressed with LPS or 5-FU. Dysregulation of hematopoietic cell fate decisions can lead to the initiation of hematopoietic malignancies like acute myeloid leukemia (AML). AML is the second most common type of leukemia but accounts for the highest number of leukemia-related deaths. Through analyzing FBXO21 expression in adult and pediatric AML samples from publicly available datasets, we identified a reduction in FBXO21 expression across AML subtypes. However, patients with a higher expression of FBXO21 had a poorer rate of survival, and patient-derived AML cell lines exhibit a higher FBXO21 protein abundance compared to BM from healthy individuals. When we knocked down FBXO21 in the patient-derived cell line, MOLM-13, cells exhibited a decrease in the number of colonies formed along with an increase in the neutrophil cell surface marker, CD15+. Our studies of FBXO21 in steady-state, stressed, and malignant hematopoiesis indicate that FBXO21 plays an important role in HSC self-renewal and quiescence. Further investigation into the role of FBXO21 could expand the current known molecular mechanisms that regulate hematopoietic lineage specification and stem cell maintenance