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.

Subtype-specific and co-occurring genetic alterations in B-cell non-Hodgkin lymphoma.

B-cell non-Hodgkin lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL, such as diffuse large B-cell lymphoma, have been comprehensively interrogated at the genomic level, but rarer subtypes, such as mantle cell lymphoma, remain less extensively characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth, including diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumors from each subtype, including frequent genetic deregulation of the ubiquitin proteasome system. In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

Ctdp1 deficiency leads to early embryonic lethality in mice and defects in cell cycle progression in MEFs.

RNA polymerase II subunit A Carboxy-Terminal Domain Phosphatase 1 (CTDP1), a member of the haloacid dehalogenase superfamily phosphatases, has a defined role in transcriptional regulation, but emerging evidence suggests an expanded functional repertoire in the cell cycle and DNA damage response. In humans, a splice site mutation in CTDP1 gives rise to the rare Congenital Cataracts Facial Dysmorphism and Neuropathy syndrome, and recent evidence from our lab indicates CTDP1 is required for breast cancer growth and proliferation. To explore the physiological function of CTDP1 in a mammalian system, we generated a conditional Ctdp1 knockout mouse model by insertion of loxP sites upstream of exon 3 and downstream of exon 4. Biallelic deletion of Ctdp1 results in lethality before embryonic day 7.5, with morphological features indicating embryo cell death and resorption. However, Ctdp1+/- mice are haplosufficient for phenotypic traits including body weight, hematological parameters, exploratory and locomotive functions. To investigate the potential mechanisms of the embryonic death caused by biallelic Ctdp1 knockout, mouse embryonic fibroblasts (MEFs) were established from Ctdp1+/+ and Ctdp1flox/flox mice. Lentivirus delivered Cre-mediated biallelic deletion of Ctdp1 in MEFs results in cell death preceded by impaired proliferation characterized by an increase in G1- and G2-phase populations and a reduction in the S-phase population. These cell cycle alterations caused by deletion of Ctdp1 are associated with an increase in p27 protein expression and a decrease in phosphorylated RB, phosphorylated Histone H3, and Cyclin B expression. Together, these results reveal that Ctdp1 plays an essential role in early mouse embryo development and cell growth and survival in part by regulating the cell cycle.

Regulation of Normal and Malignant Hematopoiesis by FBOX Ubiquitin E3 Ligases.

Hematopoiesis is responsible for numerous functions, ranging from oxygen transportation to host defense, to injury repair. This process of hematopoiesis is maintained throughout life by hematopoietic stem cells and requires a controlled balance between self-renewal, differentiation, and quiescence. Disrupting this balance can result in hematopoietic malignancies, including anemia, immune deficiency, leukemia, and lymphoma. Recent work has shown that FBOX E3 ligases, a substrate recognition component of the ubiquitin proteasome system (UPS), have an integral role in maintaining this balance. In this review, we detail how FBOX proteins target specific proteins for degradation to regulate hematopoiesis through cell processes, such as cell cycle, development, and apoptosis.

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.

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.

The CUL4-DDB1 ubiquitin ligase complex controls adult and embryonic stem cell differentiation and homeostasis.

Little is known on post-transcriptional regulation of adult and embryonic stem cell maintenance and differentiation. Here we characterize the role of Ddb1, a component of the CUL4-DDB1 ubiquitin ligase complex. Ddb1 is highly expressed in multipotent hematopoietic progenitors and its deletion leads to abrogation of both adult and fetal hematopoiesis, targeting specifically transiently amplifying progenitor subsets. However, Ddb1 deletion in non-dividing lymphocytes has no discernible phenotypes. Ddb1 silencing activates Trp53 pathway and leads to significant effects on cell cycle progression and rapid apoptosis. The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of Trp53. Conversely, depletion of DDB1 in embryonic stem cell (ESC) leads to differentiation albeit negative effects on cell cycle and apoptosis. Mass spectrometry reveals differing protein interactions between DDB1 and distinct DCAFs, the substrate recognizing components of the E3 complex, between cell types. Our studies identify CUL4-DDB1 complex as a novel post-translational regulator of stem and progenitor maintenance and differentiation.

Regulation of c-Myc ubiquitination controls chronic myelogenous leukemia initiation and progression.

The molecular mechanisms regulating leukemia-initiating cell (LIC) function are of important clinical significance. We use chronic myelogenous leukemia (CML) as a model of LIC-dependent malignancy and identify the interaction between the ubiquitin ligase Fbw7 and its substrate c-Myc as a regulator of LIC homeostasis. Deletion of Fbw7 leads to c-Myc overexpression, p53-dependent LIC-specific apoptosis, and the eventual inhibition of tumor progression. A decrease of either c-Myc protein levels or attenuation of the p53 response rescues LIC activity and disease progression. Further experiments showed that Fbw7 expression is required for survival and maintenance of human CML LIC. These studies identify a ubiquitin ligase:substrate pair regulating LIC activity, suggesting that targeting of the Fbw7:c-Myc axis is an attractive therapy target in refractory CML.

Glypican-3-mediated inhibition of CD26 by TFPI: a novel mechanism in hematopoietic stem cell homing and maintenance.

Directional migration determines hematopoietic stem/progenitor cell (HSPC) homing, which depends upon the interaction between the chemokine CXCL12 and its receptor CXCR4. CD26 is a widely expressed membrane-bound ectopeptidase that cleaves CXCL12 thereby depleting its chemokine activity. We identified tissue-factor pathway inhibitor (TFPI) as a biological inhibitor of CD26 in murine and human HSPCs. We observed low-level TFPI expression in endothelial cells in the bone marrow (BM), which did not increase following radiation injury. Treatment of HSPCs with TFPI in vitro led to enhanced HSPC migration toward CXCL12, as well as homing and engraftment in the BM upon transplantation. We found that Glypican-3 (GPC3), a heparan sulfate proteoglycan expressed on murine as well as human HSPCs, mediated this effect. TFPI did not affect CD26 activity, migration, or homing of GPC3(-/-) HSPCs, while it affected GPC1(-/-) HSPCs similar to wild-type HSPCs. Moreover, proliferation of GPC3(-/-) but not GPC1(-/-) BM HSPCs was significantly increased, which was associated with a decrease in the primitive HSC pool in BM and an increase in proportion of the circulating HSPCs in the peripheral blood. Hence, we present a novel role for TFPI and GPC3 in regulating HSC homing as well as retention in the BM.

A novel role of BMP4 in adult hematopoietic stem and progenitor cell homing via Smad independent regulation of integrin-α4 expression.

UNLABELLED: Although it is well established that BMP4 plays an important role in the development of hematopoietic system, it is less well understood whether BMP4 affects adult hematopoiesis and how. Here, we describe a novel mechanism by which BMP4 regulates homing of murine as well as human hematopoietic stem/progenitor cells (HSPCs). BMP4 treatment of murine BM derived c-kitLin-Sca-1 (KLS) and CD150CD48-KLS cells for up to 5 days in vitro prevented the culture-induced loss of Integrin-α4 (ITGA4) expression as well as homing. The effect on ITGA4 expression in response to BMP4 is mediated via SMAD-independent phosphorylation of p38 MAPK, which activates microphthalmia-associated transcription factor (MITF), known to induce ITGA4 expression. Elevated ITGA4 expression significantly enhanced HSPC attachment to bone marrow stromal cells, homing and long-term engraftment of the BMP4 treated cells compared with the cells cultured without BMP4. BMP4 also induced expression of ITGA4 on human BM derived Lin-CD34 cells in culture, which was associated with improved homing potential. Thus, BMP4 prevents culture-induced loss of ITGA4 expression on HSPCs in a SMAD-independent manner, resulting in improved homing of cultured HSPCs and subsequent hematopoietic reconstitution. KEY POINTS: Cytokine-induced loss of murine as well as human HSPC homing during ex vivo culture can be prevented by addition of BMP4. In HSPCs, BMP4 directly regulates Integrin-α4 expression through SMAD-independent p38 MAPK-mediated signaling.

Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system.

Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.

Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line.

OBJECTIVE: The microenvironment wherein hematopoietic stem cells (HSC) reside orchestrates HSC self-renewal vs. differentiation decisions. Stromal cells derived from ontogenically divergent hematopoietic microenvironments can support HSC in vitro and have been used to decipher factors that influence HSC fate decisions. Employing stromal cell lines derived from the aorta-gonad-mesonephros and embryonic liver, we aim to identify secreted factors that maintain/expand HSC in vitro. MATERIALS AND METHODS: We cultured murine lineage antigen-negative (Lin(-)) bone marrow cells in transwells above the UG26-1B6, urogenital ridge-, and EL08-1D2, embryonic liver-derived cell lines. We, also, performed real-time quantitative PCR analysis to identify differentially expressed genes from the Wnt family of proteins in ontogenically different stromal cell lines. RESULTS: Lin(-) murine bone marrow cells maintained for 3 weeks in transwells above UG26-1B6 but not EL08-1D2 cells contained competitive repopulating HSC. Addition of as few as 25% UG26-1B6 cells to EL08-1D2 feeders led to maintenance of HSC in noncontact cultures, validating soluble factors are secreted by the UG26-1B6 cells. As we found that Wnt5a was significantly higher expressed in UG26-1B6 than EL08-1D2 cells, we added Wnt5a to EL08-1D2 transwell cultures or an antibody against Wnt5a to UG26-1B6 transwell cultures. Addition of Wnt5a to EL08-1D2 transwell cultures restored maintenance of HSC, whereas addition of an anti-Wnt5a antibody to UG26-1B6 transwell cultures inhibited maintenance of competitive repopulating HSC. CONCLUSIONS: We demonstrate that stromal cell lines generated from embryonic microenvironments provide a tool to identify secreted proteins that play a role in the maintenance of HSC, and that at least one of the factors produced by UG26-1B6 cells responsible for preserving HSC is Wnt5a.

Regulation of hematopoietic stem cell differentiation by a single ubiquitin ligase-substrate complex.

Hematopoietic stem cell (HSC) differentiation is regulated by cell-intrinsic and cell-extrinsic cues. In addition to transcriptional regulation, post-translational regulation may also control HSC differentiation. To test this hypothesis, we visualized the ubiquitin-regulated protein stability of a single transcription factor, c-Myc. The stability of c-Myc protein was indicative of HSC quiescence, and c-Myc protein abundance was controlled by the ubiquitin ligase Fbw7. Fine changes in the stability of c-Myc protein regulated the HSC gene-expression signature. Using whole-genome genomic approaches, we identified specific regulators of HSC function directly controlled by c-Myc binding; however, adult HSCs and embryonic stem cells sensed and interpreted c-Myc-regulated gene expression in distinct ways. Our studies show that a ubiquitin ligase-substrate pair can orchestrate the molecular program of HSC differentiation.

Isolation and characterization of a novel population of progenitor cells from unmanipulated rat liver.

Widespread use of liver transplantation in the treatment of hepatic diseases is restricted by the limited availability of donated organs. One potential solution to this problem would be isolation and propagation of liver progenitor cells or stem cells. Here, we report on the isolation of a novel progenitor cell population from unmanipulated (that is, no prior exposure to chemicals and no injury) adult rat liver. Rat liver cells were cultured following a protocol developed in our laboratory to generate a unique progenitor cell population called liver-derived progenitor cells (LDPCs). LDPCs were analyzed by fluorescence-activated cell sorting, real-time polymerase chain reaction (RT-PCR), immunostaining and microarray gene expression. LDPCs were also differentiated into hepatocytes and biliary epithelium in vitro and examined for mature hepatic markers and urea and albumin production. These analyses showed that, LDPCs expressed stem cell markers such as cluster domain (CD)45, CD34, c-kit, and Thy 1, similar to hematopoietic stem cells, as well as endodermal/hepatic markers such as hepatocyte nuclear factor (HNF)3beta, hematopoietically-expressed homeobox gene-1, c-met, and transthyretin. LDPCs were negative for OV-6, cytokeratins (CKs), albumin, and HNF1alpha. The microarray gene expression profile demonstrated that they showed some similarities to known liver progenitor/stem cells such as oval cells. In addition, LDPCs differentiated into functional hepatocytes in vitro as shown by albumin expression and urea production. In conclusion, LDPCs are a population of unique liver progenitors that can be generated from unmanipulated adult liver, which makes them potentially useful for clinical applications, especially for cell transplantation in the treatment of liver diseases.

Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells.

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10(3)-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

Gene transfer via nucleofection into adult and embryonic stem cells.

The use of embryonic and adult stem cells as therapeutic agents is gaining momentum. A major impediment in the use of stem cells for genetic disorders is their ability to undergo genetic modification. The recognition of various site-specific integration methods open up a new avenue for gene therapy in stem cells. However, this necessitates efficient delivery of DNA molecule into cells. Most commercially used liposome-mediated transfection reagents are toxic or work poorly with stem cells. Electroporation, while effective in transfecting stem cells, is rather harsh and leads to excessive cell death. Nucleofection, a technology by Amaxa, uses a combination of electric pulse in an appropriate media, which decreases the toxicity and promotes efficient transfection of stem cells. Various types of adult and embryonic stem cells can be successfully transfected using this method, as described in this chapter.