Ubiquitin E3 Ligase FBXO9 Regulates Pluripotency by Targeting DPPA5 for Ubiquitylation and Degradation.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have unique characteristics where they can both contribute to all three germ layers in vivo and self-renewal indefinitely in vitro. Post-translational modifications of proteins, particularly by the ubiquitin proteasome system (UPS), control cell pluripotency, self-renewal, and differentiation. A significant number of UPS members (mainly ubiquitin ligases) regulate pluripotency and influence ESC differentiation with key elements of the ESC pluripotency network (including the "master" regulators NANOG and OCT4) being controlled by ubiquitination. To further understand the role of the UPS in pluripotency, we performed an RNAi screen during induction of cellular reprogramming and have identified FBXO9 as a novel regulator of pluripotency associated protein DPPA5. Our findings indicate that FBXO9 silencing facilitates the induction of pluripotency through decreased proteasomal degradation of DPPA5. These findings identify FBXO9 as a key regulator of pluripotency.

UBR5 HECT domain mutations identified in mantle cell lymphoma control maturation of B cells.

Coordination of a number of molecular mechanisms including transcription, alternative splicing, and class switch recombination are required to facilitate development, activation, and survival of B cells. Disruption of these pathways can result in malignant transformation. Recently, next-generation sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients including mutations in the ubiquitin E3 ligase UBR5. Approximately 18% of MCL patients were found to have mutations in UBR5, with the majority of mutations within the HECT domain of the protein that can accept and transfer ubiquitin molecules to the substrate. Determining if UBR5 controls the maturation of B cells is important to fully understand malignant transformation to MCL. To elucidate the role of UBR5 in B-cell maturation and activation, we generated a conditional mutant disrupting UBR5's C-terminal HECT domain. Loss of the UBR5 HECT domain leads to a block in maturation of B cells in the spleen and upregulation of proteins associated with messenger RNA splicing via the spliceosome. Our studies reveal a novel role of UBR5 in B-cell maturation by stabilization of spliceosome components during B-cell development and suggests UBR5 mutations play a role in MCL transformation.

FBXO21 mediated degradation of p85α regulates proliferation and survival of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by clonal expansion of myeloid blasts in the bone marrow (BM). Despite advances in therapy, the prognosis for AML patients remains poor, and there is a need to identify novel molecular pathways regulating tumor cell survival and proliferation. F-box ubiquitin E3 ligase, FBXO21, has low expression in AML, but expression correlates with survival in AML patients and patients with higher expression have poorer outcomes. Silencing FBXO21 in human-derived AML cell lines and primary patient samples leads to differentiation, inhibition of tumor progression, and sensitization to chemotherapy agents. Additionally, knockdown of FBXO21 leads to up-regulation of cytokine signaling pathways. Through a mass spectrometry-based proteomic analysis of FBXO21 in AML, we identified that FBXO21 ubiquitylates p85α, a regulatory subunit of the phosphoinositide 3-kinase (PI3K) pathway, for degradation resulting in decreased PI3K signaling, dimerization of free p85α and ERK activation. These findings reveal the ubiquitin E3 ligase, FBXO21, plays a critical role in regulating AML pathogenesis, specifically through alterations in PI3K via regulation of p85α protein stability.

Ubiquitin E3 ligase FBXO21 regulates cytokine-mediated signaling pathways, but is dispensable for steady-state hematopoiesis.

Hematopoietic cell fate decisions such as self-renewal and differentiation are highly regulated through multiple molecular pathways. One pathway, the ubiquitin proteasome system (UPS), controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. Ubiquitin E3 ligases serve as the substrate-recognition component of the UPS. By investigating the FBOX family of E3 ligases, we discovered that Fbxo21 was highly expressed in the hematopoietic stem and progenitor cell (HSPC) population, and exhibited low to no expression in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self-renewal, and differentiation, we generated shRNAs against FBXO21 and a hematopoiesis-specific Fbxo21 conditional knockout (cKO) mouse model. We found that silencing FBXO21 in HSPCs led to a loss in colony formation and an increase in cell differentiation in vitro. Additionally, stressing the HSPC populations in our Fbxo21 cKO mouse with 5-fluorouracil injections resulted in a decrease in survival, despite these populations exhibiting minimal alterations during steady-state hematopoiesis. Although FBXO21 has previously been proposed to regulate cytokine signaling via ASK and p38, our results indicate that depletion of FBXO21 led to altered ERK signaling in vitro. Together, these findings suggest ubiquitin E3 ligase FBXO21 regulates HSPCs through cytokine-mediated pathways.

Multi-omics reveals mitochondrial metabolism proteins susceptible for drug discovery in AML.

Acute myeloid leukemia (AML) is a devastating cancer affecting the hematopoietic system. Previous research has relied on RNA sequencing and microarray techniques to study the downstream effects of genomic alterations. While these studies have proven efficacious, they fail to capture the changes that occur at the proteomic level. To interrogate the effect of protein expression alterations in AML, we performed a quantitative mass spectrometry in parallel with RNAseq analysis using AML mouse models. These combined results identified 34 proteins whose expression was upregulated in AML tumors, but strikingly, were unaltered at the transcriptional level. Here we focus on mitochondrial electron transfer proteins ETFA and ETFB. Silencing of ETFA and ETFB led to increased mitochondrial activity, mitochondrial stress, and apoptosis in AML cells, but had little to no effect on normal human CD34+ cells. These studies identify a set of proteins that have not previously been associated with leukemia and may ultimately serve as potential targets for therapeutic manipulation to hinder AML progression and help contribute to our understanding of the disease.

From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme.

Heme is a ubiquitous and essential iron containing metallo-organic cofactor required for virtually all aerobic life. Heme synthesis is initiated and completed in mitochondria, followed by certain covalent modifications and/or its delivery to apo-hemoproteins residing throughout the cell. While the biochemical aspects of heme biosynthetic reactions are well understood, the trafficking of newly synthesized heme-a highly reactive and inherently toxic compound-and its subsequent delivery to target proteins remain far from clear. In this review, we summarize current knowledge about heme biosynthesis and trafficking within and outside of the mitochondria.

Loss of FBXO9 Enhances Proteasome Activity and Promotes Aggressiveness in Acute Myeloid Leukemia.

The hematopoietic system is maintained throughout life by stem cells that are capable of differentiating into all hematopoietic lineages. An intimate balance between self-renewal, differentiation, and quiescence is required to maintain hematopoiesis and disruption of this balance can result in malignant transformation. FBXO9, the substrate recognition component from the SCF E3 ubiquitin ligase family, is downregulated in patients with acute myeloid leukemia (AML) compared to healthy bone marrow, and this downregulation is particularly evident in patients with inv(16) AML. To study FBXO9 in malignant hematopoiesis, we generated a conditional knockout mouse model using a novel CRISPR/Cas9 strategy. Deletion of Fbxo9 in the murine hematopoietic system showed no adverse effects on stem and progenitor cell function but in AML lead to markedly accelerated and aggressive leukemia development in mice with inv(16). Not only did Fbxo9 play a role in leukemia initiation but it also functioned to maintain AML activity and promote disease progression. Quantitative mass spectrometry from primary tumors reveals tumors lacking Fbxo9 highly express proteins associated with metastasis and invasion as well as components of the ubiquitin proteasome system. We confirmed that the loss of FBXO9 leads to increased proteasome activity and tumors cells were more sensitive to in vitro proteasome inhibition with bortezomib, suggesting that FBXO9 expression may predict patients' response to bortezomib.