The delicate balance between the synthesis and the degradation of proteins ensures cellular homeostasis. Proteases act in an irreversible manner and therefore have to be strictly regulated. The ubiquitin–proteasome system (UPS) is a major pathway for the proteolytic degradation of cellular proteins. As dysregulation of the UPS is observed in most cancers including leukemia, the UPS is a valid target for therapeutic intervention strategies. Ubiquitin-ligases selectively bind substrates to target them for poly-ubiquitinylation and proteasomal degradation. Therefore, pharmacological modulation of these proteins could allow a specific level of control. Increasing evidence accumulates that ubiquitin-ligases termed mammalian seven in absentia homologs (SIAHs) are not only critical for the pathogenesis of solid tumors but also for leukemogenesis. However, the relevance and therapeutic potential of SIAH-dependent processes has not been fully elucidated. Here, we summarize functions of SIAH ubiquitin-ligases in leukemias, how they select leukemia-relevant substrates for proteasomal degradation, and how the expression and activity of SIAH1 and SIAH2 can be modulated in vivo. We also discuss that epigenetic drugs belonging to the group of histone deacetylase inhibitors induce SIAH-dependent proteasomal degradation to accelerate the turnover of leukemogenic proteins. In addition, our review highlights potential areas for future research on SIAH proteins.
Homeostasis relies on a balance between de novo protein synthesis, protein modifications, and precise degradation of misfolded and potentially harmful proteins.1, 2, 3 This cellular balance is often referred to as a ‘yin-yang’ and unobstructed cellular functions are only achievable when harmonious.
The ubiquitin–proteasome system (UPS) is the main proteolytic system of eukaryotic cells.1, 2 It is controlled by the hierarchical actions of three classes of enzymes (E1-E2-E3), of which two E1s, >30 E2 ubiquitin-conjugases, and hundreds of E3 ubiquitin-ligases have been isolated from eukaryotic cells.4 After their activation by adenosine triphosphate, ubiquitin molecules (76 amino acids, ∼8.5 kDa) are transferred to E1 enzymes. E2 ubiquitin-conjugases then accept ubiquitin moieties to covalently attach them to E3 ubiquitin-ligases or to lysine residues in substrates bound by E3s.1, 2 By interacting with defined sets of substrates and ubiquitin-conjugases, ubiquitin-ligases ensure the specificity of the UPS.4 Covalent tagging of multiple ubiquitin polypeptides connected via lysine-48 allows the recognition of substrates by the 19S subunit of the proteasome. Proteasome-associated deubiquitinylation and adenosine triphosphate-dependent translocation into the 20S core complex then leads to the cleavage of unfolded substrates by trypsin-, chymotrypsin- and caspase-like peptidases.1, 2, 3 In contrast to poly-ubiquitinylation, mono-ubiquitinylation or ubiquitinylation linked via lysine-63 of ubiquitin rather modifies protein functions and interactions.3, 4
Cell cycle progression, proliferation, senescence, differentiation, apoptosis, and a multitude of other physiologically relevant functions are controlled by ubiquitinylation-dependent proteasomal degradation.1, 2 Accordingly, disturbed proteasomal degradation rates are causative for the development and progression of malignancies.5 Thus, several ubiquitin-ligases with tumor-relevant targets appear as putative candidates for pharmacological intervention strategies.1, 2, 3 Benefits of such drugs may rely on (i) blocked proteasomal degradation and stabilization of tumor suppressors or (ii) accelerated oncoprotein elimination via induction of proteasomal degradation. Proteasomal inhibitors are currently tested or given regularly in clinics. An example is bortezomib, which is an approved drug against relapsed multiple myeloma and mantle cell lymphoma.6, 7 Further anti-cancer agents also target the proteasomal machinery. For example, histone deacetylase inhibitors (HDACi) are epigenetic drugs that induce protein acetylation and alter the cellular transcriptome to accelerate the turnover of leukemogenic proteins.
Recent reviews focus on the role of SIAHs in solid tumors and during hypoxia resulting from the disequilibrium between oxygen supply and consumption.10, 11 The main aspect of our review is to summarize the increasingly recognized functions of SIAH proteins in leukemic cells and we highlight areas for future research on these regulators of ubiquitin-dependent proteolysis.
Structure of SIAHs and mechanism of substrate recognition
SIAH proteins are highly active ubiquitin-ligases that function as scaffolds to transfer ubiquitin bound to an ubiquitin-conjugase to a substrate. The really interesting new gene (RING) zinc finger-type domain in the N-terminal part of SIAHs binds two zinc cations and makes tandem contacts with ubiquitin-conjugases. Although the SIAH C-terminal domain usually acts as the substrate-binding domain, some substrates bind to or near the RING domain.10
Human SIAH1 (∼32 kDa) has two murine homologs, Siah1a and Siah1b. These are 97% homologous with each other and 90% with SIAH1. SIAH2 (∼37 kDa) shares 85.7% amino acid homology and 68.8% identity with SIAH1, and sequence deviations mainly occur in their N-termini.10 Despite these similarities, SIAH proteins can have specific substrates (Table 1). Cancer-relevant functions of SIAH1 are influenced by SIAH isoforms. For example, the SIAH1 splice variant SIAH1S inhibits SIAH1 and promotes chemotherapy resistance and breast tumorigenesis.14 SIAH1S has so far not been reported in leukemias. A 49-amino-acid shorter form of SIAH2 (∼32 kDa, Δ23–71) has been detected in human bone marrow,12 and this protein is a highly active ubiquitin ligase.12, 15, 16
SIAHs act alone or are an essential part of multi-protein ubiquitin-ligase complexes encompassing SIAH1. These can contain the SIAH-interacting protein/Calcyclin-binding protein (SIP/CACYBP) and the adapters SKP1 and TBL1/EBI. SIP is suggested to position substrates and E2 enzymes for SIAH1-dependent poly-ubiquitinylation.10, 17 Interestingly, SIP has critical relevance for lymphoid development and cell growth.18, 19 A further adapter for SIAH1 is the zinc finger protein plenty of SH3-domains (POSH), which accentuates pro-apoptotic effects of SIAH1.20 Many direct interaction partners of SIAHs share the peptide sequence PxAxVxP (VxP, core sequence; x, any amino acid). Mutational analyses show that this so called SIAH ‘degron’ (abbreviation for ‘degradation on’) is a consensus binding motif for SIAHs and hence critical for proteasomal degradation.10, 14, 17, 21, 22
Regulation of SIAH expression and activity
In the murine myeloblastic cell line M1 (generated from a spontaneous leukemia), expression of a stably introduced temperature-sensitive mutant of the tumor suppressor p53 activates the Siah1b gene.23 In aggressive human breast cancers, expression of SIAH2 also correlates with p53 expression but as well with increased Siah2 copy numbers.24 It should be noted that the cellular context determines whether p53 increases expression of SIAHs, and these do not generally determine p53-dependent signaling.25 The transcription factor E2F1 controls cell cycle entry and apoptosis, and also binds the SIAH1 promoter to activate transcription of SIAH1 in human lung cancer cells.26 The sex hormone estrogen (17β-estradiol) induces SIAH2 at the mRNA and protein levels in human breast cancer cells.27 Surprisingly, it is still unclear whether these transcription factors control SIAH expression in cells of hematopoietic origin.
Due to self-ubiquitinylation, SIAH proteins are very unstable and often hardly detectable. Homo- and heterodimerization control the steady-state levels of SIAHs, and SIAH2 promotes proteasomal degradation of SIAH1.10, 28 Self-degradation of SIAHs is often a marker for their ability to act as ubiquitin-ligases. The deubiquitinylating enzyme USP13,29 and Vitamin K3,30 limit SIAH autodegradation and thereby its activity in kidney-derived cells and melanomas.
Further physiological and pharmacological stimuli can modulate SIAH functions (Table 2). Genotoxic drugs and hypoxia modulate the expression and activities of SIAHs p53 dependently and independently.11, 23, 24, 31, 32, 33, 34 DNA damage triggers an inhibitory phosphorylation of SIAH1 at serine-19 that is catalyzed by the kinases ATM/ATR.32, 33 The tumor suppressive kinase HIPK2 phosphorylates SIAH2 at the N-terminal serines-26,-28,-68. These inactivating phosphorylation events stabilize SIAH2 and equally disrupt its interactions with HIPK2, which itself is targeted by SIAHs.31 Tyrosine phosphorylation of SIAH1 can be induced by DNA damage. Phosphorylation of tyrosine-100,-126, activation of mixed lineage and JUN kinases, as well as POSH stabilize SIAH1.20
Targeting SIAHs to defined intracellular domains can specifically alter their activity toward substrates. For example, the stress-inducible p38 kinase promotes phosphorylation of SIAH2 at threonine-24 and serine-29 and a phospho-mimic mutant of SIAH2 localizes to perinuclear regions.34 Moreover, the nuclear export of SIAH2 is sensitive to leptomycin B (which blocks the export protein CRM1/exportin-1),34 and it is plausible that such mechanisms are altered in certain cells. For most of the above mentioned mechanisms further experimental testing is required to see whether they equally operate in blood cells and whether different chemotherapies induce distinct relevant post-translational modifications of SIAHs.
Activities of SIAHs are also controlled by the abundance of E2 ubiquitin conjugases. SIAH2 preferentially interacts with the E2 UBCH8 and SIAH1 cooperates with UBCH8 and E2s of the UBCH5 family.13, 16 Of note, HDACi induce UBCH8 to accelerate the proteasomal degradation of oncoproteins and to eliminate leukemic cells.1, 15, 16, 35, 36 Moreover, HDACi induce an accumulation of SIAHs35 (and our unpublished data). Details on the control of leukemogenic factors by HDACi are summarized in the following sections.
SIAHs and tumorigenesis
Knockout animals can provide valuable insights into protein functions. Mice lacking both Siah1a and Siah2 are not viable, but animals with individual knockouts are available. Siah1a knockout mice are severely growth retarded and often die early, with poor bone formation and twice as much osteoclasts as their wild-type counterparts.10 Siah2 null mice appear rather normal, but they present an expansion of myeloid precursors in the bone marrow suggesting a myeloproliferative phenotype. This effect particularly occurs in cells sensitive to macrophage colony-stimulating factor, a cell type which can differentiate into osteoclasts.37 These findings indicate that SIAHs modulate the immune system and this can critically determine tumorigenesis. Furthermore, they support to investigate the physiological relevance of SIAHs in the hematopoietic system.
Notably, SIAH1 can form part of a molecular signature for tumor reversion in leukemias and other tumors. In such rare anti-malignant programs, cancer cells lose their transformed state, which stems from chromosomal instability, translocations, oncogene addiction and tumor suppressor inactivation.11, 23, 38 Expression of the multidrug resistance protein MDR1 is a general problem in cancer treatment. Remarkably, SIAH1 reduces transcription of MDR1 by activating JUN kinase and binding of the transcription factor JUN to the MDR1 promoter.39 These data demonstrate that SIAHs exert crucial functions apart from the induction of proteasomal degradation.
These data, together with several reports summarized below, suggest that SIAH1 functions as a tumor suppressor. Nevertheless, recent evidence argues for a pro-tumorigenic role of SIAHs, and particularly for SIAH2, in solid tumors. For example, Siah2 deficiency attenuates the formation of aggressive prostate carcinomas in mice and SIAH2 expression correlates with lung tumorigenesis.10, 11, 40 Moreover, outcompeting SIAHs with small peptides reduces breast cancer and melanoma growth and spread.10 However, the biological consequences of SIAH inactivation in normal tissues of adult animals are unknown. Data from primary tumors show that SIAH1 locates to a region frequently lost in tumors. Allelic loss at 16q12.1 encompassing SIAH1 has, for example, been reported for hepatocellular carcinomas.10, 41 Inactivating mutations of SIAH1 have so far only been identified in gastric cancers,42 and data on putative deletions or mutations of SIAHs in leukemias are not available yet.
We will now discuss leukemia-relevant targets of SIAH1 and SIAH2, and how molecular mechanisms involving SIAH-dependent proteasomal degradation operate in leukemic cells.
Leukemia fusion proteins
AF4 and AF4/MLL
One of the most frequent genetic aberrations of the human mixed lineage leukemia (MLL) gene is the chromosomal translocation t(4;11)(q21;q23) involving ALL1 fused gene on chromosome-4 (AF4; four-eleven-leukemia, FEL).43 This abnormality is recurrently found in acute leukemia patients and results in AF4/MLL and MLL/AF4 (derivative 11/derivative 4-leukemia; Figure 1a). In t(4;11) leukemia, one fusion derivate is proteolytically cleaved by the protease threonine-aspartase-1 (Taspase1). This limited proteolysis stabilizes the SIAH-containing complex and traps SIAHs.43, 44, 45
Genetic irregularities of MLL are critical steps in the malignant transformation of hematopoietic precursors to acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). MLL encodes a histone methyltransferase ensuring proper cell cycle transitions and precise expression of developmentally important homeobox genes.43, 46, 47 The ubiquitously expressed multi-domain protein MLL is processed into an N-terminal MLLN (p300) and a C-terminal MLLC fragment (p180) by Taspase1. These form heterodimers via internal FRYC/FRYN domains (Figure 1a), to ensure stable steady-state levels and the correct subnuclear localization of MLL.48 The resulting mature MLLN320/C180 heterodimers tri-methylate histone H3 via their C-terminal SET domains to influence gene expression,43 and they assemble protein supercomplexes.49
Chromosomal translocations of the human MLL gene are a hallmark of aggressive pediatric and adult leukemias with rather poor prognosis.50, 51 In the majority of leukemia cells carrying MLL translocations, wild-type MLL is still expressed. Apparently, the fusion proteins exert dominant disease mechanisms and as such represent promising targets.43, 49 MLL-AF4 was postulated to be the oncogene in t(4;11) leukemogenesis when it was discovered.48 However, 80% of cases with translocations involving AF4 also carry AF4-MLL and nearly all established t(4;11) cell lines express both rearranged alleles.43 Indeed, recent evidence shows that AF4-MLL drives cellular transformation and leukemia induction, whereas MLL-AF4 rather disturbs cell cycle control and apoptosis.52 The absence of the AF4-MLL allele in some leukemic cells might be explained by the fact that many oncoproteins work based on a ‘hit-and-run mechanism’; AF4-MLL might be required during disease onset but is lost or even dispensable due to secondary aberrations.12, 43
To execute their (patho)biological functions, the 500 kDa precursor of MLL as well as its oncogenic MLL-fusions have to be processed proteolytically (Figure 1).48, 53, 54 Taspase1 cleaves substrates in trans by recognizing a conserved peptide motif (Q3[F,I,L,V]2D1↓G1′x2′D3′D4′).48, 54 Following autoproteolysis of the Taspase1 zymogen (cis-cleavage), its subunits assemble into an αβ-heterodimer possibly representing the active ‘monomeric’ protease (Figure 1a). Mutation of the catalytic nucleophile, threonine-234, inactivates Taspase1 in cis and in trans.55 Albeit Taspase1 localizes predominantly in the nucleus, its interaction with NPM1 allows transient access of the protease to the cytoplasm.56 MLL processing is expected to occur in the cytoplasm or during nuclear import, when MLL/Taspase1-complexes are rapidly imported into the nucleus via importins.54, 56
AF4 and AF4-MLL share the first three exons of the AF4 gene. These exons encode 360 amino acids comprising the N-terminal half of the ALF domain including the SIAH-dependent degron (Figure 1), which allows an exceptionally tight regulation that precludes AF4 protein accumulation in non-transformed cells.12 Accordingly, mutating crucial amino acid residues within this motif stabilizes AF4 in vivo, and this short portion of AF4 can bind SIAHs and exerts oncogenic activity when transported to the nucleus.44, 45 In the physiological situation, the unprocessed pathological AF4-MLL fusion protein still interacts with SIAHs to become subjected to proteasomal degradation. Similar to MLL, AF4-MLL is processed by Taspase1. Upon cleavage by Taspase1, the AF4-MLL.N and MLL.C cleavage products become protected and the complex including SIAHs and AF4-MLL is stabilized. This molecular mechanism is supposed to trap SIAHs,12 and to consequently disturb steady-state protein levels relying on SIAH activity against tumor-relevant proteins.32 Chemico-genetic targeting of Taspase1 would thus not only counteract oncogenic AF4-MLL accumulation but also SIAH trapping and disturbed proteasomal homeostasis. However, Taspase1 is not affected by available protease inhibitors and no genetic trans-dominant-negative mutants are available yet.55, 57 Although all consequences of the SIAH/AF4-MLL interaction remain to be understood, these data demonstrate a crucial role for SIAHs in leukemogenesis driven by AF4-MLL. In light of such findings, it is surprising that nothing is known about a putative effect of SIAHs on the stability of Taspase1 and vice versa.
Remarkably, a recent report revealed that HDACi evoke cell death of t(4;11)-positive primary infant acute lymphoblastic leukemia cells, and downmodulation of MLL-AF4 on the mRNA and protein levels was observed in the t(4;11)-positive precursor B cell acute lymphoblastic leukemia line SEM.58 It is possible that the induction of UBCH8-dependent SIAH functions attenuates AF4-MLL and it is likewise interesting to see whether HDACi may affect Taspase1. Extrapolating our current knowledge, one could expect synergistic therapeutic benefits from rationally designed combination regimen. Applying HDACi with still to be identified Taspase1 inhibitors should prevent AF4-MLL stabilization and consequently accelerate its SIAH-dependent degradation. As aberrant recruitment of the methyltransferase hDOT1L to MLL via the AF10 part alters histone H3 methylation at K79 and activates gene expression promoting leukemogenesis, drugs blocking DOT1L may also be given with HDACi to correct aberrant gene expression driven by MLL fusions and DOT1L.59
AML1-ETO and PML-RARα
The transcription factor acute myeloid leukemia-1 (AML1) and the core-binding factor-β (CBFβ) form the transcriptional activator CBF. Aberrations of AML1 are judged as characteristic initiating events in leukemogenesis.60 The AML1-ETO fusion originates from a balanced translocation involving AML1 and the gene encoding the transcriptional repressor eight-twenty-one (ETO). AML carrying this translocation t(8;21)(q22;q22) accounts for 5–12% of AML cases, which renders it the most common translocation reported in AMLs.61 In AML1-ETO, nearly the entire open reading frame of ETO fuses with the runt homology domain of AML1, which controls AML1 target genes ensuring proper hematopoietic differentiation. Consequently, AML1-ETO represses differentiation genes by recruiting transcriptional repressors, histone deacetylases and associated corepressors, via its ETO portion.5, 60 Besides the aberrant AML1-ETO-mediated gene repression, AML1-ETO blocks other transcription factors, such as C/EBPα, c-JUN, PLZF, SMAD3, VDR and p53.62 Curiously, a splice variant of AML1-ETO, AML1-ETO9a lacking its C-terminal NHR3/NHR4 domains recruiting HDACs and corepressors, has been isolated from human AML cells. This protein turned out to be more oncogenic than AML1-ETO itself in murine models.63
Aberrant repression of genes inducing differentiation is also linked to the development of acute promyelocyctic leukemia. Acute promyelocyctic leukemia represents 5–8% of all AML cases and is characterized by the PML-RARα protein, which results from a fusion between the promyelocytic leukemia (PML) and retinoic acid receptor-α (RARα) gene (t(15;17)(q22;q21)). PML-RARα disturbs RAR target gene expression promoting maturation of the granulocytic lineage. Albeit stimulation with retinoic acid releases corepressors from the RARα/RXR heterodimer, the oncogenic PML-RARα recruits corepressor complexes and DNA methylating enzymes that resist physiological levels of retinoic acid.5, 64 Moreover, PML suppresses tumor growth by facilitating the acetylation and stabilization of p53 and this property is lost in PML-RARα.5, 62, 64 Data from murine models suggest that PML-RARα requires additional genetic changes to induce aggressive acute promyelocyctic leukemia.65, 66 Although it is clear that chromosomal translocations found in AML can encode transcription factors disrupting proper hematopoietic transcription, PML-RARα is yet the only such oncogenic fusion protein that can be targeted specifically. Acute promyelocyctic leukemia patients have a favorable prognosis as they respond to pharmacological doses of retinoic acid-mediating dissociation of corepressor complexes from PML-RARα, followed by its proteasomal and caspase-dependent degradation.5, 16, 62
Several reports demonstrate that HDACi kill AML1-ETO-positive cells in vitro,8, 35, 67, 68, 69 and HDACi preferentially induce degradation of AML1-ETO and PML-RARα over the intact proteins.35, 69, 71 The underlying mechanism might be that AML1-ETO and PML-RARα are preferred targets for proteasomal degradation induced by UBCH8 in conjunction with SIAH1 and SIAH2 (Figure 2a).16, 35, 72 For PML-RARα and PML, the coiled-coil domain was identified as the structural determinant for SIAH-mediated proteasomal degradation.72 PML-RARα hence represents an example for a target not dependent on the SIAH degron (Figure 2b). Furthermore, SIAHs supersede the stabilization of PML-RARα by the putative ubiquitin-ligase TRIAD1.16 It is not absolutely clear why UBCH8 and SIAH prefer AML1-ETO over AML1 and ETO as a substrate. Resolving this question might allow to specifically target the oncoprotein. Although not proven experimentally, the occurrence of four SIAH recognition motifs in AML1-ETO and only two in AML1 and ETO suggests that this molecular feature attracts SIAHs to AML1-ETO (Figure 2b). Functionally relevant dimerization and oligomerization of fusion proteins5, 61, 73 allows the clustering of SIAH degrons and this may further concentrate SIAHs proximal to AML1-ETO and PML-RARα. Such a mechanism might equally explain why SIAH1 is a nuclear protein in AML1-ETO- or PML-RARα-positive leukemic cells, albeit SIAH1 occurs in the cytosol or dispersed throughout the cell in cells lacking these fusions.15, 16, 35 Of note, similar findings were made for SIAH1 and BOB.1.74 This B cell-specific coactivator interacts with SIAHs in B and T cells and evokes redistribution of SIAH1 to the nuclear compartment.74 These observations are also reminiscent of the trapping of SIAHs by AF4-MLL (Figure 1b); AML1-ETO and PML-RARα may disturb protein degradation by titrating them away from other cancer-relevant substrates.
It is equally thinkable that wild-type proteins and leukemia fusion proteins are differentially degraded in the presence of HDACi because they variably depend on the HSP90 chaperone facilitating protein folding and stability. HDACi were reported to induce an inhibitory acetylation of HSP90 to liberate oncogenic proteins for proteasomal degradation.75, 76 However, HDACi targeting class I HDACs (HDACs1,-2,-3,-8) can reduce leukemia-relevant proteins, although they cannot affect HSP90, which is specifically deacetylated by the class IIa deacetylase HDAC6.35, 76, 77 It is plausible that both HSP90 inhibition and induction of enzymes of the UPS including UBCH8-SIAH1/-SIAH2 operate in a concerted manner.
Although the above data suggest HDACi as a strategy against AML1-ETO-positive cells, acetylation of lysine-43 by the acetyltransferase p300 can promote oncogenesis driven by AML1-ETO9a.78 Acetylation-dependent activation of this oncoprotein though contrasts previous data and the anti-leukemic effects of HDACi toward AML1-ETO-positive cells.8, 35, 61, 67, 68, 69, 70 Nonetheless, the HDACi-inducible proteasomal degradation of AML1-ETO may trump acetylation-dependent effects. One can also speculate that acetylation of AML1-ETO and of other proteins is required in certain tumor stages and dispensable or even damaging in others. It is equally possible that HDACi affect p300 in AML1-ETO-positive cells, and hyperacetylation of AML1-ETO in HDACi-treated cells might trigger its destabilization.
Another open question is the putative role of p53 for the proteasomal degradation of AML1-ETO and PML-RARα. Although it is known that both factors antagonize p53,5, 62, 64 which can induce SIAH1 and SIAH2 in certain cells,23, 24, 25 it is unclear whether leukemia fusion proteins attenuate their proteasomal turnover by inactivating the p53 pathway. Interestingly, AML1-ETO-expressing cells are also sensitive to corticosteroids, which like HDACi evoke a proteasome-dependent depletion of AML1-ETO.80 SIAH-dependent proteasomal degradation could also eliminate the more oncogenic AML1-ETO9a63 and treatment-resistant PML-RARα,81 as long as they still contain SIAH recognition motifs (Figure 2). This might, for example, be the case for arsenic trioxide-resistant PML-RARα mutants harboring mutations in the PML B2 domain preceding the coiled coil.81 Such notions support the usage of HDACi to increase the success of chemotherapies and to prevent the advent of leukemic clones with additional aberrations in leukemia fusion proteins. Further leukemia fusion proteins might also be sensitive to the HDACi-induced UBCH8-SIAH module; an example could be PAX5-PML containing the PML coiled coil.82 One should though keep in mind that not any leukemia fusion protein is a target of SIAHs, for example, STAT5-RARα (17q21.1-q21.2) is not,35 and not all binding partners of SIAHs become degraded by proteasomes.16, 83 Such findings illustrate the specificity of the SIAH-UPS-axis.
FLT3 mutants and further signaling molecules
FMS-like tyrosine kinase-3 (FLT3), which crucially controls hematopoiesis, belongs to the type III receptor tyrosine kinases subfamily, also including c-KIT, c-FMS and PDGF-receptors.84 FLT3 contributes to the proliferation, differentiation and survival of hematopoietic stem cells. In normal bone marrow, FLT3 is mainly expressed in early CD34-positive myeloid and lymphoid progenitors and its expression ceases during differentiation.85
Activating mutations of FLT3 tie in with poor clinical outcome and occur in 20–30% of AML patients. The most common FLT3 mutations in AML are internal tandem duplications (ITDs).86 These locate to the regulatory juxtamembrane region, which regulates the access of adenosine triphosphate to the kinase domain. Small fractions of patients suffering from chronic myeloid leukemia, myelodysplasia and ALLs also carry FLT3 mutants.85 FLT3-ITDs show ligand-independent dimerization and cause cytokine-independent receptor activation promoting cell proliferation linked to STAT5 activation and malignancy.87, 88 FLT3-ITD is sufficient to induce a myeloproliferative phenotype and in some cases also lymphoid disease in transgenic or bone marrow-transplanted mice.86
As FLT3-ITD is a disease-associated ‘driver’ mutation, it is a valid therapeutic target.86 Drugs targeting constitutively active FLT3 undergo extensive clinical testing and AC220 is currently the most specific inhibitor for the catalytic activity of FLT3.86, 88, 89 Unfortunately, FLT3-ITD-positive AML patients develop resistance to AC220 under therapy.86, 89 FLT3 inhibitor development therefore aims to target drug-resistant FLT3-ITD mutants.86, 90, 91 It appears promising that the clinically used pan-HDACi LBH589 was found to enhance the proteasomal elimination of FLT3-ITD at therapeutically relevant concentrations.15 SIAH1 and SIAH2, in conjunction with the HDACi-inducible UBCH8, catalyze poly-ubiquitinylation and proteasomal degradation of FLT3-ITD (Figure 2a).15 This effect of SIAHs toward FLT3-ITD is specific and not evoked by the ubiquitin-ligases HDM2, c-CBL and RLIM.15 Apparently, SIAHs recognize FLT3-ITD dependent on its tyrosine phosphorylation (Figure 2b), which indicates that they particularly eliminate mutant and not wild-type FLT3 ensuring hematopoiesis. Future research will clarify how SIAHs, which have no known motif for binding phosphorylated proteins, recognize tyrosine phosphorylated FLT3 and whether this is valid for all FLT3 mutants. It may be that this ability of SIAHs is conferred by interaction partners within SIAH complexes.10, 14 Independent of these details, HDACi-inducible UBCH8-SIAH-axes should target phosphorylated FLT3 mutants whether they are resistant to TKi or not. Accordingly, HDACi act as very potent drugs in combination with TKi.15, 76, 88
Interestingly, expression of FLT3 is controlled by factors being regulated by SIAHs. For example, the FLT3 promoter is directly controlled by MLL-AF4 and MLL fusion-positive leukemias express high levels of FLT3 mRNA.43, 92 Thus, MLL-AF4 might accelerate leukemogenesis in the context of mutated FLT3. Mutagens and cooperating lesions such as FLT3-ITD also collaborate with AML1-ETO and PML-RARα in inducing AML.5, 65 Hence, SIAHs seem to have bifurcating antagonistic effects on class I mutations halting proper differentiation and class II mutations conferring proliferation and survival. Thus, HDACi inducing UBCH8 and SIAHs may be particularly useful against such diseases (Figure 2a).15, 35, 62
Clearly, it is worth investigating if additional transcription factors and kinases are controlled by SIAHs and can be pharmacologically controlled. For example, p95vav/VAV1 (a hematopoietic proto-oncogene activated by IGF/IGF-1R), which is required for antigen-dependent signaling in lymphocytes and subject to control by SIAH2,83 may be sensitive to HDACi. Curiously, in Siah2−/− mice no regulation of TRAF2, VAV1 and OBF.1 was found, while these animals presented an expansion of myeloid progenitors.37 It should be noted that interaction of SIAH2 with VAV1 does not accelerate its proteasomal degradation but blocks its signaling to JUN kinase and the transcription factors AP1 and NFAT. This mechanism requires the N-terminus of SIAH2 and might be linked to T-cell receptor (TCR) stimulation and interleukin-2 expression.83 Further interesting candidate molecules that are putatively affected by HDACi comprise the BCL2-associated athano-gene-1 (BAG1) and the transmembrane receptor DCC. Both molecules are targets of SIAHs.10, 11 BAG1 is required to sustain expression of the anti-apoptotic factors BCL2, BCL-XL, MCL1 in human AML cell lines,93 and DCC is a prognostic factor in AML. As DCC loss is associated with worse prognosis, its elimination by SIAHs might be undesired.94
As tyrosine phosphorylation allows FLT3-ITD to associate with the UBCH8-SIAH module (Figure 2b), such phosphorylation-dependent recognition might affect further leukemogenic kinases, such as constitutively active JAK2V617F,75 or BCR-ABL (t(9;22)(q34;q11)).95 This idea is supported by synergistic effects of HDACi with drugs antagonizing JAK2 or BCR-ABL.75, 96, 97 Signaling via the rat sarcoma proto-oncogene (RAS) and conditions evoking hypoxia are also regulated by SIAHs. Vitamin K3 is a specific inhibitor of SIAH2 (Table 2), and prolyl hydroxylase PHD3 (which controls hypoxic signaling) and Sprouty2 (a regulator of RAS) are stabilized and melanoma formation in vitamin K3 treated mice becomes attenuated.30 This finding might have relevance for the treatment of leukemias, as RAS signaling can determine the success of chemotherapy.98 Hypoxic signaling promotes pro-tumorigenic gene expression and the development of treatment-resistant cancers. Hypoxic conditions may contribute to the pathogenesis of multiple myeloma, a malignant plasma cell disorder.99 The proteasomal inhibitor bortezomib is used against this incurable disease,7 and SIAH-dependent proteolysis may affect multiple myeloma development.
The pan-HDACi AR-42 is currently in phase II clinical trials for the treatment of relapsed or recurrent hematological and solid cancers. It was recently found that this agent shuts down constitutively active mutants of KIT in spontaneously developed primary canine mast cell tumors.77 Thus, KIT might be controlled via the UBCH8-SIAH-axis. Because AML1-ETO increases expression of KIT and KIT mutations are frequently found in CBF-AML patients (including t(8;21) and inv(16));100 HDACi could attack such leukemic cells via both, targeting Kit as well as AML1-ETO.
Transcription and elongation factors
Data on leukemia fusion proteins show that aberrant transcriptional repression involving HDACs is associated with cancer. The leukemia-relevant transcriptional co-repressor SKI represses retinoic acid signaling by stabilizing HDAC3. SKI binds HDAC3 and SIAH2 to block the ubiquitin-ligase activity of SIAH2.101 Remarkably, SKI can act as an oncoprotein via its interaction with the lineage-specific transcription factor PU.1. PU.1-dependent transcription stimulates the terminal differentiation of myeloid cells into macrophages, and loss of PU.1 is linked to myeloid leukemia.102 As SKI represses PU.1 by recruiting HDAC3, stabilization of HDAC3 at differentiation-inducing promoters by blocking SIAH2101 may equally contribute to leukemogenesis (Figure 3). Interestingly, the SKI-dependent SIAH2-HDAC3 module may be controlled by non-coding microRNAs (miRs) in AML with aberrant chromosome-7 (-7/del7q). These show upregulation of SKI and loss of miR-29a on chromosome 7q.103 Further analyses are needed to judge whether miR-29a modulates SKI and SIAH2 to antagonize the development of this highly aggressive AML.
HDAC3 is also relevant for the pathogenesis of DLBCLs (diffuse large B cell lymphomas), which can be divided into three subtypes. DLBCLs arrested during plasmacytic differentiation have activated gene expression profiles and form the ABC subgroup. These tumors originate from post-germinal center B cells and are more frequently therapy resistant than germinal center B cell lymphomas.104 Curiously, ABC lymphomas have higher HDAC3 levels, constitutive phosphorylation of the cancer-relevant transcription factor STAT3 and expression of its anti-apoptotic target MCL1. Although STAT3 activation increases chemotherapy resistance,105 it also confers sensitivity of ABC tumors to the HDACi LBH589.106 It will be interesting to see whether upregulation of HDAC3 in certain DLBCLs associates with dysregulation of SIAHs.
The transcriptional corepressors NCOR and SMRT control definitive erythropoiesis and myeloid lineage commitment.107 FLT3 is negatively regulated by NCOR in acute monoblastic/monocytic leukemia (5–10% of adult AMLs). In turn, activation of FLT3 triggers loss of NCOR, and its ablation potentiates FLT3 ligand-induced proliferation. Thus, therapeutic restoration of NCOR may be a therapeutic strategy.108 However, caution is required as differentiation genes are suppressed by NCOR/SMRT/HDAC-complexes associating with leukemia fusion proteins.8 As NCOR tightly interacts with HDAC3 and both are controlled by SKI and SIAH in adherent cells,107, 109 SIAHs might relieve aberrant transcriptional repression exerted by leukemic fusions (Figures 2a and 3).
Several additional molecules control NCOR/HDAC3-dependent transcriptional control. TBL1, its homologs GPS2 and TBLR1 interact with HDAC3, NCOR and SMRT.107 This complex mediates corepressor/coactivator exchange reactions by a ligand-dependent recruitment of the 19S proteasome.107 In macrophages, NCOR controls c-JUN/c-FOS-dependent networks regulating inflammation, migration and collagen catabolism. Phosphorylation of c-JUN induces removal of NCOR/HDAC3/TBL1/TBLR1 complexes by ubiquitinylation complexes containing SIAH2.110 Therefore, it is plausible that SIAH2-dependent proteolysis is critical for myeloid differentiation and inflammation.
BOB.1/OBF.1 is a coactivator for OCT transcription factors and critical for B cell development and germinal center formation.74 IgM-crosslinking of the human germinal center-like Burkitt’s lymphoma line Ramos increases SIAH1 and BOB.1 mRNA expression, but decreases the levels of BOB.1 (Table 2). Furthermore, in primary tonsilar B cells expression of the activation marker CD38 correlates with low SIAH1 mRNA levels.74 From these data it was proposed that rapidly dividing germinal center centroblasts lose B cell receptor expression and BOB.1 accumulates. It is unclear whether the interaction of SIAH2 with BOB.1 affects its stability and whether SIAHs antagonize B cell proliferation via BOB.1.
The transcription factor β-catenin is a target of SIAH1 and SIAH2,17, 18, 19, 111 and SIP1 is involved in the p53-inducible SIAH1-mediated degradation of β-catenin.112 Of note, SIP1−/− thymocytes fail to fully engage TCR-β gene rearrangements and have increased apoptosis rates resulting in reduced thymic cellularity.18 Furthermore, β-catenin signaling is required for stemness and the oncogene-induced transformation of progenitor cells (for example, via MLL-AF9),113 and the E2F1-dependent induction of SIAH1 suppresses β-catenin signaling.26 In B cell lymphomas carrying Epstein-Bar-virus, hexachlorophene (Table 2) antagonizes repression of SIAH1 by the viral latent membrane protein-1.33, 114 SIAH1 induced by this chemical is active against β-catenin, and consequently expression of its target genes cyclin D1 and c-MYC is reduced and lymphoma growth is halted p53-independently.114
Interestingly, SIAH1 and SIP1 are also linked to metabolic changes. Both are involved in the proteasome-dependent attenuation of cytoplasmic amounts of the cyclin-dependent kinase inhibitor p27 upon glucose deprivation. In addition, compared with wild-type cells SIP−/− embryonic fibroblasts have augmented cytosolic p27 linked to increased motility and invasiveness.19 Like SIAH1, SIAH2 can affect the cellular metabolism. SIAH2 promotes proteasomal degradation of cytosolic levels of the dihydrolipoamide S-succinyltransferase, which is a subunit of the mitochondrial α-ketoglutarate dehydrogenase complex catalyzing the conversion of α-ketoglutarate to succinyl-CoA.115 This rate-limiting step in the ‘Krebs’/TCA/citric-acid cycle allows the generation of energy equivalents and intermediate molecules. Cancer cells rather produce energy by glycolysis than via mitochondrial oxidation (‘Warburg’ effect).
Super elongation complexes (SECs) promote transcriptional elongation stimulating normal and leukemia-associated gene expression. Together with MLL, SECs instruct homeotic gene expression for pattern formation and development.46 Accordingly, several more or less frequent translocation partners of MLL coexist in SECs, which contain the transcription elongation factors P-TEFb, ELL1/ELL2, and the scaffold proteins AFF4 and AF4/AFF1.46 ELL2 is a stoichiometrically limiting protein of SECs and is specifically targeted for ubiquitin-mediated degradation by SIAH1, but not by SIAH2.13 Depending on the dose of SIAH1, the scaffolding factor AFF4 has the ability to protect ELL2 by attenuating the access of SIAH1. AFF4 can though also be subjected to proteasomal degradation induced by SIAH1.13 This mechanism illustrates the complex regulatory patterns that govern whether SIAHs can degrade a protein. This SIAH1-dependent control of transcriptional elongation processes can be influenced by small molecules. The plant-derived phorbol and protein kinase C activator prostratin (from the mamala tree bark) as well as the hybrid polar compound hexamethylene bisacetamide (HMBA; Table 2) enhance accumulation of the transcription elongation factor eleven-nineteen lysine-rich leukemia (ELL2) and thereby formation of SECs. Mechanistically, prostratin and hexamethylene bisacetamide decrease expression of SIAH1 mRNA and protein levels and reduce ELL2 poly-ubiquitinylation and proteasomal degradation. This pathway is discussed as a possible mechanism to promote HIV transcription for the elimination of latent viral reservoirs.13
Interestingly, sequence variations in the 36 amino acid RING domains of SIAH1 and SIAH2 are responsible for their different abilities to induce degradation of ELL2. Of three non-conserved exchanges SIAH2 alanine-96 versus SIAH1 serine-88 and SIAH2 glutamine-106 versus SIAH1 proline-98 appear critical for their variable effects toward ELL2.13 These differences do not affect the interaction of SIAH2 with ELL2, but its ability to induce ELL2 degradation. Nonetheless, a lack of cofactors for SIAH2 in HeLa cervix carcinoma cells might also be the reason for the failure of SIAH2 to induce degradation of ELL2.13 As SIAH2 preferentially interacts with UBCH8,13 and as HDACi induce UBCH8,8, 16 these agents may render SIAH2 active against ELL2 to modulate oncogenic transcription. Dimerization of SIAHs and the SIAH2-dependent degradation of SIAH110, 28 could further complicate this scenario.
Future studies will likely reveal that additional proteins are controlled by UBCH8-SIAH-dependent proteasomal pathways and hence susceptible to HDACi-induced proteasomal degradation. Candidates might be the transcription factors LMO2 and WT1. Overexpression of LMO2 is linked to T-ALL and undergoes proteasomal degradation by a yet unidentified pathway.116 WT1 is a pan-leukemic marker that is controlled by the HDACi-dependent induction of UBCH8 and by the serine protease HTRA2/OMI,117, 118 and these processes may involve SIAHs. Furthermore, HDACi alter immune and inflammatory processes, for example, via the forkhead transcription factor FOXP3, which controls regulatory T cell subsets.119 It still always has to be noted that not each protein interacting with SIAHs becomes degraded by the proteasome and that SIAHs can exert functions beyond the induction of ubiquitin-dependent proteolysis.13, 16, 39, 74
Modulation of cancer-relevant signaling by small molecules is highly desired and still accepted as the ‘gold standard’ in the clinics. Recent (pre)clinical research revealed novel opportunities to directly and indirectly modulate proteasomal networks. Current research aims to identify which ubiquitin-ligases target specific substrates for poly-ubiquitinylation and proteasomal degradation, and how these enzymes can be controlled.
From the data available we hypothesize that SIAHs are druggable enzymes that are relevant for cancer development and during therapy. Although the SIAH substrate binding domain may be a useful structure to block these enzymes,11 SIAHs can eliminate leukemia fusion proteins, aberrant kinases and chemotherapy resistance proteins.1, 12, 13, 15, 16, 35, 39, 71, 72, 107 All these factors are crucial for leukemogenesis and therapy, and this supports the view that SIAHs may antagonize leukemogenesis. Enhanced SIAH activities can be achieved by regulating the abundance of an ubiquitin-conjugase as in the case of HDACi treatment.1, 15, 16, 35, 36 Moreover, the tremendous potential of exploiting aberrations in the UPS as an Achilles heel to attack tumor cells may set the avenue for developing novel combination therapies.15, 75, 76, 88, 96, 120
However, caution is equally required, as particularly SIAH2 appears to promote cell growth, metastasis and the hypoxic drive of solid tumors.10, 11, 30, 31, 32 Additional data including the analysis of primary blood cancers are necessary to further understand the role(s) of SIAHs for leukemogenesis. The next goal is then to analyze the biological and functional consequences of the SIAH-substrate relationship and how it can be exploited for pharmacological control and the benefit of patients.
As summarized in this review, SIAH ubiquitin-ligases represent master regulators destroying leukemogenic proteins via intricate protease and kinase signaling pathways. We speculate that SIAHs, likely in conjunction with UBCH8, are scavengers that accelerate the proteasomally mediated elimination of rogue leukemia-relevant proteins.1 The basal and induced levels of UBCH8 and SIAHs may predict responsiveness and therapy success for cases in which a leukemogenic driver is sensitive to HDACi and UBCH8-SIAH-dependent proteasomal degradation. The loss of oncoproteins may likewise be a valid pharmacodynamic marker.62
Collectively, increasing evidence supports a central role of SIAH proteins for cancer biology. Understanding and exploiting the functional impact of SIAHs in leukemia, in liaison with newly designed and increasingly specific drugs, may pave the avenue for new rational therapeutic concepts to fight malignancies.
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We wish to thank all members of our groups and Professor T Heinzel for helpful discussions and fruitful collaboration. Our research is supported by grants from the Deutsche Krebshilfe, the Deutsche Forschungsgemeinschaft, and the Wilhelm-Sander-Stiftung. We apologize for works not cited due to space limitations or an oversight on our part.
OHK performed bibliographic search, analyzed data, and wrote the paper. SKK, RHS, JH and GB commented on the article and wrote parts of the review. All authors approved the paper for publication purposes.
Gesine Bug has received honoraria and travel grants from Novartis Pharma GmbH and from Celgene GmbH. The remaining authors declare no conflict of interest.
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Krämer, O., Stauber, R., Bug, G. et al. SIAH proteins: critical roles in leukemogenesis. Leukemia 27, 792–802 (2013). https://doi.org/10.1038/leu.2012.284
- leukemia fusion protein
- proteasomal degradation
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