Regulation of normal and malignant hematopoiesis by FBOX ubiquitin E3 ligases

Abstract

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 the hematopoietic stem cell 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, play 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.

Regulation of Hematopoiesis

Hematopoiesis (see Glossary) is responsible for key functions required for survival including oxygen transportation, host defense, and injury repair in vertebrates. This is maintained throughout life by hematopoietic stem cells (HSC).[1] HSCs are the self-renewing population capable of generating all hematopoietic lineages. Within the bone marrow (BM), HSCs are mainly quiescent but can be signaled to replenish the hematopoietic system in times of stress, whereas progenitor populations have extensive proliferation potential to replenish rapidly turned over hematopoietic populations.[2] An intricate balance between self-renewal, differentiation, and quiescence is required to maintain hematopoiesis. Disruption of this balance can lead to anemia, immune deficiencies, as well as leukemia or lymphomas.

HSCs give rise to multi-potent progenitor (MPP) populations that differentiate to progeny with more restricted differentiation potential. The two subclasses of progenitor populations are the common myeloid progenitor (CMP) and the common lymphoid progenitor (CLP).[3, 4] The CMP gives rise to all myeloid cells that include granulocytes, monocytes, macrophages, erythrocytes, megakaryocytes, mast cells, and certain antigen presenting dendritic cells (DC), whereas the CLPs give rise to mature cells required for innate and adaptive immunity, including T-cells, B-cells, NK cells, and certain DC subsets.[5] Both intrinsic and extrinsic molecular mechanisms including the microenvironment, cytokine signaling, and transcription factors play a key role in specifying hematopoietic differentiation. The ubiquitin proteasome system (UPS), a key modulator of protein stability and function, regulates cell fate decisions, thus adding an additional molecular mechanism regulating hematopoiesis.[6] Ubiquitin E3 ligases are the substrate-recognizing component of the UPS that target specific proteins, tag substrates with polyubiquitin chains, and promote their proteasome-mediated degradation (Box 1). E3 ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a lysine residue within a specific set of substrate proteins; in the case of UPS targeted degradation, they produce a K48-linked poly-ubiquitin chain (Figure 1).[7] Since E3 ligases determine substrate ubiquitin modification, in humans there are over 600 known E3 ligases each recognizing a specific set of substrates.[8] E3 ubiquitin ligases can be classified as homologous to the E6-AP carboxyl terminus (HECT) domain, really interesting new gene (RING)-finger, or RING-between-RING (RBR) based on their domains and mode of transferring ubiquitin to the substrate.[9] Although extensive studies have addressed the importance of microenvironment and transcriptional regulation in hematopoietic differentiation, recent studies have identified a novel role of ubiquitin E3 ligases in hematopoietic homeostasis and malignant transformation. Here, we discuss recent work elucidating the roles of FBOX E3 ligases that play a crucial role in normal and malignant hematopoiesis, and their potential future use in combating malignancies, immune system regeneration, and replenishing hematopoietic populations following stress.

Box1; Ubiquitin Proteasome System Regulation.

The UPS coordinates degradation of proteins globally and compartmentally within a cell and is a key regulatory mechanism for many cellular processes including cell cycle progression, proliferation, development, and differentiation.[94] This system functions through an enzymatic cascade in which the E1 ubiquitin activating enzyme activates ubiquitin, which is then conjugated to an E2 ubiquitin-conjugating enzyme and subsequently passed to a substrate protein through interaction with an E3 ubiquitin ligase. Proteins that receive a poly-ubiquitin chain are recruited to the proteasome for proteolysis (Figure 1). The key components of this system are the E3 ubiquitin ligases due to their critical role of determining substrate specificity.

Each E3 ubiquitin ligase mediates the transfer of ubiquitin molecules from E2 enzymes to a lysine residue within a specific set of substrates proteins. Since E3 ligases determine substrate ubiquitin modification, there are over 600 known or predicted E3 ligases, each recognizing a specific set of substrates. The largest family of E3 ligases is the Skp1-Cul1-FBOX (SCF) family of RING-finger E3 ligases. E3 ubiquitin ligases can be classified as homologous to the E6-AP carboxyl terminus (HECT) domain, Really Interesting New Gene (RING)-finger, or RING-between-RING (RBR) based on their domains and mode of transferring ubiquitin to the substrate. HECT E3 ligases are unique in that they bind the E2 and transfer the activated ubiquitin to a catalytic cysteine within the conserved c-terminal HECT domain which then directly transfers the ubiquitin to the substrate protein.[9] RING E3 ligases comprise the largest subset of ubiquitin ligases, containing a canonical RING finger domain, made up of multiple cysteine and histidine residues interspersed with other amino acids, that interacts with two zinc ions.[95] Unlike HECT E3 ligases, RING finger proteins act as scaffolding proteins (or protein complexes) to bring the E2 in proximity with the substrate.[9] The last class, RBR E3 ligases, acts as a combination of the other two. The first RING domain facilitates discharge of the ubiquitin from the E2 onto the second RING domain with a catalytic cysteine similar to the HECT domain.[96]

Figure 1. Steps of the Ubiquitin Proteasome System (UPS).

The first step of the UPS system is the activation of ubiquitin by the E1 ubiquitin ligase (1) by using ATP. Ubiquitin is then conjugated by the E2 ubiquitin ligase (2). The E3 ubiquitin ligase interacts with both the protein that is targeted for degradation and the E2 ubiquitin ligase aiding the transfer of ubiquitin onto the target substrate (3). The formation of the polyubiquitin tail signals for the target substrate to be sent to the proteasome (4) and later degraded (5). Ub= ubiquitin

FBOX E3 ligases in the regulation of normal and malignant hematopoiesis

The SKP1-CUL1-FBOX (SCF) family of RING-finger E3 ligases constitutes the largest group of ubiquitin E3 ligases.[10] SCF ligases are named after the elements that compose the core of the complex. S-phase kinase-associated protein 1 (SKP1), an adaptor protein, and Cullin 1 (CUL1), a scaffolding protein, bring the ubiquitin binding RING-finger protein, RBX1, in proximity with the substrate recognition FBOX protein (Figure 2). To date, 69 distinct FBOX proteins have been identified, each including normally two domains: an FBOX domain which interacts with SKP1, and a substrate-recognition domain.[11] This family of proteins is subdivided into three categories based on their substrate-recognition domain: FBXL proteins contain leucine rich repeats, FBXW proteins contain WD repeats, and FBXO proteins contain another type of substrate-recognition domain or have an unidentified protein-binding site.[12, 13] One characteristic shared by most FBOX proteins is the need for a phosphodegron (or amino acid recognition motif). Examples include DSGxxS degron motif for β-TrCP1 (FBXW1) and TPxxS degron motif for FBXW7, where the FBXW7 degron motif for its substrate c-MYC is LLPTPPLS.[14, 15] FBOX proteins can also recognize degrons that are modified by glycosylation. In the case of FBXO6, it recognizes T-cell receptor alpha-subunit (TCRα) through a glycosylated degron.[16] FBOX proteins recognize approximately 20% of proteins degraded by the UPS and can ubiquitinate substrates important for cell survival, cell cycle regulators, transcription factors, and cell-surface receptors (Figure 3).[17, 18] They regulate the expression and activity of a variety of tumor suppressors, oncogenes, and target proteins involved in the hallmark pathways of cancer, making them interesting proteins to study in normal and malignant hematopoiesis (Figure 4).[19]

Figure 2. Schematic of the SKP1-CUL1-FBOX (SCF) Complex.

The SCF Complex is primarily composed of the adaptor protein SKP1, the scaffolding protein CUL1, and the substrate recognition component, the FBOX protein. The complex also contains the ubiquitin binding RING-finger protein, RBX1, and the E2 ubiquitin ligase. The FBOX protein binds to its target substrate after it has been phosphorylated. The SCF complex allows for the transfer of ubiquitin from the E2 ubiquitin ligase onto the target substrate, producing a ubiquitin tail, and signaling the target substrate to be sent to the proteasome and degraded. Ub= ubiquitin, P= phosphate

Figure 3. FBOX proteins contribute to regulating the cell cycle in the vertebrate hematopoietic system.

Model of the cell cycle including prominent cyclin-cyclin dependent kinase (CCN-CDK) complexes as regulated by FBOX proteins either directly or via other cell cycle regulators. Activation in green, inhibition in red.

Figure 4. FBOX proteins contribute to regulating hematopoiesis in vertebrates.

Schematic of hematopoiesis including FBOX proteins known to regulate specific branches. Regulation by FBOX proteins can affect the promotion of cells through various branches of hematopoiesis as well as alter their functions, such as leading to malignancy.

Cell cycle regulation

SKP2 (FBXL1)

S-phase kinase-associated protein 2 (SKP2) has long been shown to regulate cell cycle progression by associating with the Cyclin A (CCNA)- Cyclin dependent kinase 2 (CDK2) complex and by ubiquitinating cyclin dependent kinase inhibitors (CDKNs) that prevent the G₁-S transition, including p21 and p27 in human fibroblast cells.[20, 21] SKP2 regulates hematopoietic stem and progenitor cells (HSPCs) and increases in response to myeloablative therapies, preventing inhibition of cell cycle progression and allowing HSPCs to repopulate mature populations.[22] Following myeloablative therapy, mice transplanted with Skp2^−/− BM exhibited impaired HSPC engraftment and repopulation of hematopoietic populations.[22] Contrary to reports that SKP2 facilitates BM repopulation, another group found that Skp2^−/− mice had enhanced long-term reconstitution by increasing cell cycle progression and augmenting the stem cell pool, likely by a loss of negative regulation of CCND1.[23] The authors postulate that this disparity likely stems from discrepancies in the HSC populations studied or genetic background of the mice used, though further studies are needed to determine the reasoning behind the differences. Though SKP2 primarily functions in cell cycle regulation, it can also regulate the transcription of β-catenin. Skp2^−/− mice have exhibited reduced HSC homing to and engraftment in BM and spleen due to loss of β-catenin.[24]

SKP2 is commonly highly expressed in cancers, including leukemia and lymphoma.[25] In T-cell acute lymphoblastic leukemia (T-ALL), SKP2 has been reported to drive leukemogenesis in both human patients and mouse models; however, Skp2^−/− mice present no alterations in normal T cell development.[26] Transgenic mice expressing T-cell specific SKP2 under the Cd4 promoter and co-expressing Nras develop T cell lymphomas with shorter latency, leading to decreased animal survival relative to mice expressing either Nras or SKP2 alone.[25] SKP2 expression in tumor sections from patients with diffuse large cell lymphoma (DLBCL) has correlated with high grade tumors [25], and Skp2^−/− mice are resistant to developing lymphoma induced by PTEN loss, when crossed to Pten^+/− mice.[26, 27] SKP2 activity and the regulation of cell cycle progression, particularly with regard to leukemic stem cells (LSCs) and tumor cell populations, suggests that inhibition of SKP2 might prove efficacious as a potential target for drug discovery, warranting further investigation.[23, 28] SKP2 illustrates the multifaceted role that many FBOX E3 ligases play in both normal and malignant hematopoiesis: its primary known function is to regulate cell cycle progression but additional roles target alternative substrates, allowing SKP2 to coordinate a variety of processes and signaling pathways that promote cell survival.

β-TrCP1/2 (FBXW1/FBXW11)

β-transducin repeat-containing proteins one and two (β-TrCP1/2), collectively known as β-TrCP, play a major role in cell cycle progression by targeting multiple cell cycle regulators for proteasomal degradation. β-TrCP is uniquely capable of promoting or inhibiting cell cycle progression either by degrading claspin and WEE1 G2 checkpoint kinase (activation) or CDC25A phosphatase and early mitotic inhibitor 1 (EMI1) (inactivation) in mouse embryonic fibroblasts (MEFs) isolated from β-TrCP^−/− mice and human cell lines, respectively.[29–31] β-catenin and the WNT signaling pathway are essential for fetal mouse HSPC development and provide protection from oxidative stress.[32] Overexpression of β-TrCP2 has increased proliferation in vitro and enhanced tumor development in vivo in DBA2 mice utilizing a patient derived lymphocytic leukemia cell line (L1210).[33] Moreover, A β-TrCP1^ΔFBOX mutant acted as a dominant-negative in mouse myeloma cells, prohibiting engraftment and cell survival in mice.[34] In this study, analysis of hematopoietic cells expressing β-TrCP^ΔFBOX showed they were unable to home to the BM and were consequently more likely to undergo apoptosis. Furthermore, treatment with the β-TrCP inhibitor, pyrrolidine dithiocarbamate (PDTC), reduced tumor burden in mice predisposed to developing myeloma, and increased survival (using human myeloma patient derived cell lines), suggesting a potential use of β-TrCP inhibitors in combination therapies to treat hematologic diseases for patients with increased β-TrCP.[34] Like β-TrCP, other FBOX proteins have been found to regulate the cell cycle at various phases by targeting different substrates.

FBXL12

In hematopoiesis, FBXL12 plays a role in regulating T-cell maturation and proliferation by degrading aldehyde dehydrogenase 3 (ALDH3) and cyclin dependent kinase inhibitor 1B (CDKN1B) [35]. Fbxl12 mRNA and protein are highly expressed in the mouse thymus, the primary organ for T-cell development. Fbxl12^−/− mice exhibit normal numbers of immature CD4⁺CD8⁺ T-cells but reduced numbers of single positive T-cells.[35] In this study, FBXL12 was highly expressed in CD4⁺CD8⁺ T-cells and ALDH3 amounts inversely correlated within that population.[35] This suggested that Fbxl12-mediated degradation of ALDH3 caused a block in T-cell differentiation at the double positive to single positive transition[35]. Recent studies have shown that Fbxl12 expression is regulated by pre-TCR signaling in T cells, and also mediates T-cell proliferation to induce maturation from immature double negatives to double positives in mice.[36] It appears to do so by working in concert with SKP2 to destabilize CDKN1B which promotes the proliferative burst that occurs as T-cells mature in the Lck^CRE, Fbxl12^f/f mouse model.[36] These studies suggest that FBXL12 plays a key role in regulating mouse thymocyte differentiation via ALDH3, and through cell cycle regulation.

FBXW7

The most well characterized FBOX E3 ligase in the hematopoietic system is FBXW7. FBXW7 plays a crucial role in maintaining HSCs and preventing tumorigenesis by regulating quiescence.[37–42] FBXW7 is known to contribute to ubiquitination-mediated degradation of c-MYC, NOTCH, Cyclin E (CCNE), and MCL1.[43–46] Conditional Fbxw7^f/f mice crossed to inducible Mx-1Cre⁺, cause its deletion in all hematopoietic cells, leading to increased proliferation of HSCs following depletion, resulting in premature depletion of the normal HSC population.[38, 39] Utilizing Fbxw7^f/f Mx-1Cre⁺, Fbxw7^f/f was crossed to the cMYC^GFP mouse model determining that the regulation of HSCs occurs primarily through c-MYC.[47] HSC quiescence is maintained in the absence of c-MYC resulting in inhibiting cell cycle initiation.[47, 48] FBXW7 also regulates cell cycle by targeting CCNE, the primary cyclin responsible for coordinating S-G₂ transition. Knock-in mouse models of CCNE expressing both Fbxw7^T74A and Fbxw7^T393A mutations, demonstrated the inability of FBXW7 to interact with and ubiquitinate CCNE. Dysregulation of CCNE in mice led to anemia and red blood cell differentiation defects.[49] Of note, increased CCNE expression did not impair HSC self-renewal; however, it altered the ability of the HSC to differentiate into all hematopoietic populations.[50] Together, these studies illustrate the key role of FBXW7 in regulating mouse HSC maintenance and differentiation.

From another angle, multiple cancer-related FBXW7 mutations have been shown to occur in human carcinomas, including T-ALL.[51–53] Most of these mutations affect three arginine residues within the WD40 protein-binding domain and inhibit binding to phosphorylated substrates.[15] Noteworthy, FBXW7 mutations and NOTCH mutations in the phosphodegron PEST domain are mutually exclusive.[54] T-cell specific Fbxw7^f/f crossed to CD4^CRE mice develop thymic lymphoma due in part to excessive accumulation of MYC.[55] Additionally, BM knockout of Fbxw7^f/f, CD4^CRE also develop T-ALL within 16 weeks in mice.[56] FBXW7 mutations identified in T-ALL patients result in the formation of oncogenic mutants that lead to increased tumor formation in FBXW7^D527G and FBXW7^D510E acute T-cell leukemia xenografts in mice.[42]

The tumor suppressive nature and ability to regulate HSC quiescence make FBXW7 an interesting protein for studying LSCs – a rare, quiescent subpopulation necessary for leukemia cell propagation.[57] A major impediment to leukemia eradication stems from the inability of current therapies to target LSCs, as conventional chemotherapy toxicity requires cellular proliferation. FBXW7 regulation of LSC quiescence has been studied in T-ALL and chronic myeloid leukemia (CML). In a Notch1^ΔE T-ALL mouse model, loss of FBXW7 (Fbxw7^f/f, Mx-1^CRE)-mediated ubiquitination of c-MYC initiated proliferation.[40] In CML, FBXW7 had the opposite effect of stabilizing LSCs by reducing c-MYC amounts in Fbxw7^f/f, Mx-1^CRE HSPC (transduced with BCR-ABL expressing retrovirus) that were transplanted into a lethally irradiated C56BL/6 recipients. Ablation of FBXW7 initiated cycling of LSCs making them vulnerable to treatment with imatinib.[41, 57] These findings suggested that FBXW7 could acts as either an oncogene or a tumor suppressor depending the cell type, substrate, and disease.

FBXL2

FBXL2 participates in cell cycle regulation by targeting CCND2 for degradation. Unlike most FBOX proteins that recognize phosphodegrons, FBXL2 recognizes a calmodulin-binding motif and must outcompete calmodulin to interact with substrates.[58] Patients with acute leukemia have often shown increased CCND2 and decreased FBXL2 which aligns with data showing that reintroduction of FBXL2 causes G₀ arrest and apoptosis in leukemic cell lines.[58] In the case of FBXL2, further investigation focusing on upstream regulation, as opposed to substrates, may aid in the development of targeted therapies for potentially treating acute leukemia.

FBXO4

Uncontrolled proliferation, a hallmark of cancer, often occurs when mutations lead to overexpression or stabilization of cell cycle promoters such as CCND1.[59] Cell cycle progression is controlled by sequential expression of CCNs and CDKs, which are controlled by coordinated expression of E3 ubiquitin ligases that tag them for proteasomal degradation. Aberrant CCN/CDK expression results in an inappropriate halt or progression through the cell cycle that leads to disease development.[60] Under normal conditions, CCND1 is ubiquitinated for degradation by the FBXO4-αB after phosphorylation by GSK3β.[61] Fbxo4^−/− mice exhibit elevated CCND1, which lead to increased transformation of MEFs in vitro, and mice succumb to multiple tumor phenotypes, including lymphomas, due to elevated amounts of CCND1.[62] In nearly 100% of mantle cell lymphoma (MCL) patients, CCND1 is constitutively activated causing B-cell progression through the cell cycle with unchecked proliferation.[63] Although mutations have not been identified in MCL, these findings suggest that FBXO4 expression in MCL can contribute to CCND1 overexpression and MCL pathogenesis.

FBXO6

Little is known about upstream regulation of FBOX proteins, however one FBOX protein whose upstream regulation has been studied is FBXO6. Regulation of FBXO6 in t(8;21)-patient-derived leukemic cell lines occurs through MAPK signaling.[64] Depletion of the MAPK ERK2 has led to increased expression of FBXO6 in patient-derived AML cell lines.[64] The group conducting this study concluded that ERK2 expression in t(8;21) acute myeloid leukemia (AML) promoted cell proliferation by increasing cell cycle progression upon the regulation of genes including FBXO6. Indeed, FBXO6 targets the protein checkpoint kinase 1 (CHK1), another cell cycle regulator, for protein degradation.[65] CHK1 is responsible for delaying cell cycle progression by stabilizing the replication fork until DNA repair occurs and synthesis resumes.[65]

FBXO6 has also been shown to regulate the innate immune response in human cell lines.[66] Type I interferons (IFN-I) activate the innate immune response but prolonged activation can lead to autoimmune inflammatory conditions, so activation of these proteins is carefully controlled. In the 293T cell line, FBXO6 was identified as an E3 ubiquitin ligase for IFN-regulatory factor 3 (IRF3), an IFN-I regulatory protein.[66] IRF3 activity negatively regulated IFN-I signaling in human 239T IFN-β luciferase reporter cell lines, thus preventing inappropriate or excessive inflammation suggesting a potential role of FBXO6 in immune response. However, further studies are required to determine its role in anti-viral immune responses in vivo.[66]

FBXO7

Largely studied for its role in Parkinson’s disease and mitophagy, FBXO7 also has a cell cycle regulatory function by mediating the interaction of CCND, CDK6, and p27.[67] FBXO7s non-canonical, non-ubiquitinating function allows FBXO7 to regulate hematopoiesis.[68] Instead of destabilizing the CCND/CDK6 complex through ubiquitin-mediated degradation, FBXO7 assists in complex assembly.[67] In the hematopoietic system, increased expression of Fbxo7 by retroviral expression in murine HSPCs inhibited colony formation of HSPCs in a p53-dependent manner.[69] Additionally, Fbxo7^LacZ/LacZ mouse model displayed increased pro-B cell and pro-erythroblast populations by accelerating G₁ phase resulting in smaller, more abundant cells – a characteristic previously linked to cancer development.[70]

In the mouse Ba/F3 cell line, FBXO7 was also found to inversely correlate with CD43, a marker for immature B-cells, suggesting integration of cell cycle progression and regulation of differentiation, two mechanisms that have not previously been linked through a single protein.[70] In the knockout model Fbxo7^LacZ/LacZ, Fbxo7 was linked to prevention of anemia by stabilizing p27, thus causing cell cycle arrest in terminally differentiating erythroblasts, allowing them to become mature red blood cells.[68] Furthermore, overexpression by retroviral vectors in mouse HSPCs combined with p53 deletion promoted formation of cell lymphomagenesis.[69] This protein’s ability to act in a non-ubiquitination function is not unique within the FBOX family or within the subset that regulate hematopoiesis.

Non-canonical functions

KDM2B (FBXL10)

As a non-canonical component of the polycomb repressive complex one (PRC1), the main function of lysine demethylase 2B (KDM2B) is in histone H3 lysine K36 mono- and di-demethylation.[71] PRC1 alters the epigenetic landscape of CDKN promoters, decreasing CDK expression.[72] Previous work has shown that KDM2B monoubiquitinates histone H2A, a necessary modification for the recruitment of the PRC complex to DNA.[71, 73] H2A monoubiquitination may also function as an epigenetic marker for recruitment of other transcription factors.[74] KDM2B can control developmental and definitive hematopoiesis by regulating integral pathways such as NOTCH or WNT. The conditional mouse model of Kdm2b (Vav^CRE, Kdm2b^f/f) that deletes Kdm2b during hematopoietic development demonstrates loss of Kdm2b leading to impaired HSPC function and altered proliferation.[75] Moreover, AML, T-ALL, and B-cell acute lymphoid leukemia (B-ALL) patient samples were have shown high KDM2B expression that correlated with increased HSPC proliferation and malignant transformation.[76]

KDM2B has also been shown to induce transcriptional repression resulting in increased proliferation in Myelodysplastic syndrome (MDS).[77] Perhaps most interesting in the context of MDS, murine HSCs with forced Kdm2b overexpression maintained their self-renewal capabilities compared to normal endogenous amounts, linking high Kdm2b expression with increased proliferative potential – a hallmark of cancer.[72] A mouse model with Kdm2b transgene inserted into the Sca-1 cassette to overexpress Kdm2b in mouse HSPCs led to the development of myeloid or B-cell leukemia with 100% penetrance, indicating that Kdm2b expression contributes to malignant transformation.[78] Additionally, knockdown using shRNA lentiviral systems to stably knockdown Kdm2b in Hoxa9-Meis1 mouse model of AML leads to decrease LSC self-renewal, a population difficult to target.[76] As a PRC complex component, KDM2B primarily functions as a histone demethylase rather than an E3 ligase, though its ubiquitination function may be necessary for complete PRC activity.[73]

KDM2A (FBXL11)

KDM2A, the paralog to KDM2B, functions similarly and demethylates H3K36. Though it does not complex with PRC1, KDM2A co-immunoprecipitates with SUZ12 (a core component of PRC2) and is concurrently recruited with PRC2 components to the human FGFR2 gene.[79] Methylation patterns vary greatly with some signatures stably maintained through mitosis and others changing throughout the organism’s lifetime. A study of pediatric peripheral blood serial collections beginning at three months of age and continuing through five years demonstrated that genes responsible for chromatin remodeling, including KDM2A and KDM2B, undergo dynamic epigenetic alterations in leukocytes through early childhood.[80] Not only is methylation important for gene expression in early childhood development, but it also acts as a regulatory mechanism of normal and malignant hematopoiesis.

Two histone methyltransferases necessary for hematopoietic development include absent, small, or homeotic discs 1 like (ASH1L) and mixed lineage leukemia (MLL) that methylate H3 at K36 and K4, respectively.[81, 82] Chromosomal translocations involving MLL often result in leukemogenesis. KDM2A, like its paralog, is a histone demethylase preferential to H3K36me2 and is the counterpart to ASH1L.[83] KDM2A overexpression opposes ASH1L activity by demethylating chromatin at MLL target genes causing reduced wild-type MLL chromatin occupancy in both human patient derived cell lines and mouse transformed MLL-AF9 HSPC.[83] Despite no effect on MLL-fusion protein chromatin occupancy, KDM2A has counteracted MLL-driven transcription and suppressed leukemic transformation. In a sleeping beauty transposon screen in a constitutively active Stat5 mouse model of B-ALL, Stat5 and Kdm2a activation cooperated to drive leukemogenesis. Expression of KDM2A inversely correlated with B-ALL patient survival.[84] The previously reported FBOX proteins have all been shown to either play a direct or indirect role in cell cycle progression within the hematopoietic system, but the remaining FBOXs discussed herein regulate normal and/or malignant hematopoiesis through other mechanisms.

Regulation of other pathways

FBXO9

Relatively little is known about the ligase function of FBXO9 within the hematopoietic system. Previous studies have shown overexpression of FBXO9 in multiple myeloma (MM) patients.[85] In MM, FBXO9 facilitated ubiquitination and degradation of telomere maintenance 2 (TEL2) and TEL2 interacting protein 1 (TTI1) enhancing malignant cell proliferation and survival.[85] Analysis of FBOX proteins in murine HPSC populations revealed high Fbxo9 expression in HSCs.[86] Deletion of Fbxo9 in early-stage murine hematopoietic cells resulted in only marginal changes in BM-derived HSPCs with a decrease in megakaryocyte erythroid progenitors (MEP).[86] Though Fbxo9 knockout does not directly cause malignant transformation, when combined with expression of the fusion protein characteristic of inv(16) AML in mice (Cbfb^+/56M Fbxo9^−/−), it led to increased AML blasts within the BM, more aggressive disease, and shorter survival time.[86] Taken together, these studies suggest a role of FBXO9 in hematopoietic development and malignant transformation; however, further studies are required to define the role of FBXO9 in hematopoiesis.

FBXL5

FBXL5 affects murine HSPC function by regulating iron homeostasis that, in turn, affects HSPCs’ abilities to engraft and reconstitute mature blood cells.[87] This study has shown that Fbxl5^Δ/Δ murine HSCs are more susceptible to oxidative stress induced by myeloablative agents and are prone to loss of quiescence and stem cell exhaustion in an iron-dependent manner.[87] MDS patients occasionally have reduced FBXL5 expression, perhaps contributing to hematopoietic failure characteristic of MDS.[87] While some FBOX family members, such as SKP2 and FBXW7, have been extensively studied and their roles in cell cycle regulation are well defined, others, like FBXL5, may have novel cellular functions such as regulating iron homeostasis.

FBXO11

Though hematopoietic differentiation begins in the BM, many stages of maturation occur in secondary hematopoietic organs. B-cells fully mature in the spleen whose germinal centers are key areas of differentiation and activation of antibody secreting plasma cells. Expression of transcriptional repressor BCL6 is necessary for proper germinal center development and inactivation is crucial for post-germinal center differentiation.[88] Mutations causing aberrant germinal center regulation and BCL6 dysregulation can lead to hematologic malignancies. BCL6 expression is switched off through multiple signaling pathways, including FBXO11-mediated proteasomal degradation in humans.[89] Similar to the phenotype observed with increased BCL6, the conditional knockout mouse model of Fbxo11 under B cell specific CRE, FBXO11^fl/fl-Cy1cre^tg/+, has enlarged germinal centers, loss of B cell homeostasis and develop lymphoma[90]. Analysis of FBXO11 highlights the important role many of these FBOX proteins play: many have been shown to be important for maintaining normal hematopoietic cell development, and expression changes, either higher or lower, which can lead to initiation and/or progression of malignancies.

FBXL13

Two leukemia-initiating genomic alterations prevalent among therapy-related leukemias are monosomy 7 and del(7q).[91] One report identified a common patient deletion in 7q22 that contained 14 known genes, including FBXL13[92]. After sequencing analysis of several leukemia samples for functional mutations in FBXL13 and epigenetic expression changes affecting mRNA production, the authors concluded the evidence was insufficient to classify FBXL13 as a novel tumor suppressor.[92] However, the inconclusive evidence does not preclude FBXL13 from hematopoietic regulation or tumor suppression, but illustrates the need for deeper investigation into mechanistic changes that occur when FBXL13 ligase activity is lost in healthy and disease states.

Concluding Remarks

As illustrated above, the UPS, through several FBOX family proteins contributes to carefully regulating hematopoiesis. While some have been shown to have a large impact on many facets of normal and malignant hematopoiesis, such as FBXW7, others have weaker correlations, such as FBXL13. Here, we commented on 15 of the 69 FBOX proteins with known functions associated with hematopoiesis. Although the functions of the remaining 54 FBOX proteins in hematopoiesis remain undetermined, it is likely that many of these proteins regulate relevant pathways. Although the majority of FBOX proteins discussed regulate cell cycle progression, recent literature demonstrates that FBOX proteins have more diverse targets and play a role in differentiation, apoptosis, and cellular metabolism.

Recent studies have impacted both basic hematology and translational research by identifying novel molecular mechanisms important for hematopoiesis and immune development and by defining mechanism contributing to anemia, leukemia, and lymphoma. Of relevance, the ubiquitin proteasome system is amenable to molecular targeting, thus providing novel putative targets for the development of therapeutics for hematopoietic malignancies. The therapeutic agent lenalidomide, used to treat both MM and MCL, was recently shown to alter ubiquitin E3 ligase CRL4^CRBN specificity for its substrates.[93] Understanding the role of FBOX proteins in both normal and malignant hematopoiesis may provide important information regarding their physiological role and potential as therapeutic targets. In the case of SKP2, depletion of Skp2 has not altered differentiation and survival of normal mouse T-cells, whereas depletion in T-ALL cells has led to apoptosis suggesting targeting SKP2 might not affect the healthy counterpart.[26] Likewise in CML, depletion of FBXW7 in LSC has led to decreased proliferation and increased apoptosis, but has had little to no effect on normal HSC.[41, 57] Together, these findings reveal specific targets for future drug discovery that might not deplete the healthy HSC population. The data further support the concept that modulating ubiquitin ligases and their substrates merit further investigation in both basic and translational research. Future studies present the opportunity of identifying novel ubiquitin-regulated substrates important for hematopoiesis, immune system regeneration, hematopoietic recovery following stress (i.e.; infection, irradiation, or chemotherapy) and HSC expansion for transplantation; they might also help unveil candidate molecules for drug discovery in the treatment of leukemias and lymphomas. The diversity of functions of FBOX proteins illustrates the importance of elucidating the role of each family member and determining their functions in both normal and malignant hematopoiesis.

Outstanding Questions.

• Could targeting FBOX proteins, upstream of regulatory proteins or FBOX target substrates, be effective in treating hematopoietic malignancies? The drug bortezomib has been used in combination with chemotherapy to treat patients with hematopoietic malignancies. This drug works by binding the 26S proteasome, leading to a block in proteasomal degradation, and has had unpredictable results across different subtypes of AML patients. However, could more targeted therapeutics display a more efficient and standard result?

• Could other FBOX proteins be important in maintaining hematopoiesis? The depth to which FBOX proteins have been investigated varies for each protein. For example, SKP2 and FBXW7 have been investigated further, while others, such as FBXL5 and FBXO9, have been understudied. This variability in investigations has rendered certain FBOX proteins, orphan proteins, with target substrates yet to be discovered.

• Why are certain FBOX proteins particularly essential for blood homeostasis and why is their function so frequently perturbed in blood cancers? Is this due to the role of FBOX proteins in cell cycle regulation?

• The recent development of proteolysis targeting chimera (PROTACs), can link an E3 ligase with a protein binding molecule and hijack the UPS system to target specific proteins. Can FBOX proteins be utilized for PROTACs in the hematopoietic system to target specific proteins relevant for blood malignancies?

Highlights.

• Recent work has shown that FBOX proteins play an integral role in maintaining the balance of quiescence, self-renewal, and differentiation of hematopoietic stem and progenitor cells (HSPC) in vertebrates.

• Altering the activity of certain key FBOX E3 ligases has been correlated with loss of quiescence, aberrant differentiation, and malignant transformation, providing key putative targets for drug discovery.

• Although FBOX proteins are known to target cell cycle regulators, new studies demonstrate their ability to regulate other key pathways in hematopoiesis, such as iron homeostasis, signal transduction, transcription, and apoptosis, by targeting specific proteins for degradation.

Glossary

Acute myeloid leukemia (AML)

hematopoietic cancer that progresses quickly leading to an excess production of immature myeloid cells and inversely, a decrease of red blood cells, platelets, and lymphoid cells. AML is characterized by recurrent genetic abnormalities.

Common lymphoid progenitor (CLP)

immature cell population that has committed to the lymphoid branch of hematopoiesis. Mature blood cells within the lymphoid branch include NK cells, B-cells, and T-cells.

Common myeloid leukemia (CML)

hematopoietic cancer that progresses slowly leading to an excess production of partially mature myeloid cells and inversely, a decrease of red blood cells, platelets, and lymphoid cells. CML is associated with the translocation of the BCR gene on chromosome 22 and the ABL gene on chromosome 9.

Common myeloid progenitor (CMP)

immature cell population that has committed to the myeloid branch of hematopoiesis. Mature blood cells within the myeloid branch include granulocytes, monocytes, erythrocytes, and platelets.

Hematopoiesis

process that continually generates all cell types in the blood. This process is maintained throughout life by the hematopoietic stem cell.

Hematopoietic stem cell (HSC)

The most immature hematopoietic population that has the capability of generating all hematopoietic cells and maintains hematopoiesis throughout life.

Leukemic stem cell (LSC)

mutated immature cell population that remains quiescent (and thus is a difficult to target with current chemotherapeutics).

Mantle cell lymphoma (MCL)

aggressive and rare type of lymphoma that develops in naïve B-cells located in the mantle zone of a lymphoid follicle, which leads to accumulation of malignant B-cells.

Myelodysplastic syndrome (MDS)

Disease that develops due to a block in the ability of immature stem cells to mature into healthy blood cells; it commonly precedes AML.

Myeloablative therapies

treatments such as high dose radiation that eradicate malignant cells but also destroy HSPC; myeloablative therapies are accompanied with subsequent HSC transplantation.

NRAS

member of the RAS family of proteins responsible for signal transduction, named for it discovery in neuroblastoma.

Phosphodegron

specific amino acid sequence, typically containing a serine or threonine, whose phosphorylation permits recognition by the E3 ligase preceding ubiquitination for proteasomal degradation.

Skp1-Cul1-FBOX (SCF)

E3 ubiquitin ligase complex that binds to targeted proteins and promotes their degradation through the UPS. The complex is composed of an adaptor protein (Skp1), a scaffolding protein (Cul1), a RING protein that allows for the binding of an E2 ubiquitin-conjugating enzyme, and the target substrate specific protein (FBOX).

T-cell acute lymphoblastic leukemia (T-ALL)

type of acute leukemia resulting in extensive lymphoblasts in the peripheral blood and BM. T-ALL is the most common childhood leukemia.

Ubiquitin proteasome system (UPS)

system that controls protein amounts by tagging them with polyubiquitin chains and promoting their degradation through the proteasome.