SIAH Proteins: Critical Roles in Leukemogenesis

REVIEW SIAH proteins: critical roles in leukemogenesis

OH Kra¨mer1, RH Stauber2, G Bug3, J Hartkamp4 and SK Knauer5

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.

Leukemia (2013) 27, 792–802; doi:10.1038/leu.2012.284 Keywords: leukemia fusion protein; proteasomal degradation; SIAH1; SIAH2; UBCH8

INTRODUCTION functions are controlled by ubiquitinylation-dependent proteaso- 1,2 Homeostasis relies on a balance between de novo protein mal degradation. Accordingly, disturbed proteasomal synthesis, protein modifications, and precise degradation of degradation rates are causative for the development and 5 misfolded and potentially harmful proteins.1–3 This cellular progression of malignancies. Thus, several ubiquitin-ligases with balance is often referred to as a ‘yin-yang’ and unobstructed tumor-relevant targets appear as putative candidates for 1–3 cellular functions are only achievable when harmonious. pharmacological intervention strategies. Benefits of such The ubiquitin–proteasome system (UPS) is the main proteolytic drugs may rely on (i) blocked proteasomal degradation and system of eukaryotic cells.1,2 It is controlled by the hierarchical stabilization of tumor suppressors or (ii) accelerated oncoprotein actions of three classes of enzymes (E1-E2-E3), of which two E1s, elimination via induction of proteasomal degradation. 430 E2 ubiquitin-conjugases, and hundreds of E3 ubiquitin- Proteasomal inhibitors are currently tested or given regularly in ligases have been isolated from eukaryotic cells.4 After their clinics. An example is bortezomib, which is an approved drug 6,7 activation by adenosine triphosphate, ubiquitin molecules (76 against relapsed multiple myeloma and mantle cell lymphoma. amino acids, B8.5 kDa) are transferred to E1 enzymes. E2 Further anti-cancer agents also target the proteasomal machinery. ubiquitin-conjugases then accept ubiquitin moieties to For example, histone deacetylase inhibitors (HDACi) are epigenetic covalently attach them to E3 ubiquitin-ligases or to lysine drugs that induce protein acetylation and alter the cellular residues in substrates bound by E3s.1,2 By interacting with transcriptome to accelerate the turnover of leukemogenic defined sets of substrates and ubiquitin-conjugases, ubiquitin- proteins. ligases ensure the specificity of the UPS.4 Covalent tagging of Recent reviews focus on the role of SIAHs in solid tumors and multiple ubiquitin polypeptides connected via lysine-48 allows the during hypoxia resulting from the disequilibrium between oxygen 10,11 recognition of substrates by the 19S subunit of the proteasome. supply and consumption. The main aspect of our review is to Proteasome-associated deubiquitinylation and adenosine summarize the increasingly recognized functions of SIAH proteins triphosphate-dependent translocation into the 20S core complex in leukemic cells and we highlight areas for future research on then leads to the cleavage of unfolded substrates by trypsin-, these regulators of ubiquitin-dependent proteolysis. chymotrypsin- and caspase-like peptidases.1–3 In contrast to poly- ubiquitinylation, mono-ubiquitinylation or ubiquitinylation linked via lysine-63 of ubiquitin rather modifies protein functions and STRUCTURE OF SIAHS AND MECHANISM OF SUBSTRATE interactions.3,4 RECOGNITION Cell cycle progression, proliferation, senescence, differentiation, SIAH proteins are highly active ubiquitin-ligases that function as apoptosis, and a multitude of other physiologically relevant scaffolds to transfer ubiquitin bound to an ubiquitin-conjugase to

1Center for Molecular Biomedicine (CMB), Department of Biochemistry, University of Jena, Jena, Germany; 2Molecular and Cellular Oncology/Mainz Screening Center (MSC), University Medical Center Mainz, Mainz, Germany; 3Department of Internal Medicine II, Hematology/Oncology, Goethe University Hospital, Frankfurt/Main, Germany; 4Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Aachen, Germany and 5Centre for Medical Biotechnology (ZMB), Department of Molecular Biology, University Duisburg-Essen, Essen, Germany. Correspondence: Dr OH Kra¨mer, Center for Molecular Biomedicine CMB, Department of Biochemistry, University of Jena, Jena, Germany. E-mail: [email protected] Received 22 August 2012; revised 14 September 2012; accepted 17 September 2012; accepted article preview online 5 October 2012; advance online publication, 23 November 2012 Emerging impact of SIAH1 and SIAH2 in leukemia OH Kra¨mer et al 793 a substrate. The really interesting new gene (RING) zinc finger-type temperature-sensitive mutant of the tumor suppressor p53 domain in the N-terminal part of SIAHs binds two zinc cations and activates the Siah1b gene.23 In aggressive human breast cancers, makes tandem contacts with ubiquitin-conjugases. Although the expression of SIAH2 also correlates with p53 expression but as SIAH C-terminal domain usually acts as the substrate-binding well with increased Siah2 copy numbers.24 It should be noted that domain, some substrates bind to or near the RING domain.10–13 the cellular context determines whether p53 increases expression Human SIAH1 (B32 kDa) has two murine homologs, Siah1a and of SIAHs, and these do not generally determine p53-dependent Siah1b. These are 97% homologous with each other and 90% with signaling.25 The transcription factor E2F1 controls cell cycle entry SIAH1. SIAH2 (B37 kDa) shares 85.7% amino acid homology and and apoptosis, and also binds the SIAH1 promoter to activate 68.8% identity with SIAH1, and sequence deviations mainly occur transcription of SIAH1 in human lung cancer cells.26 The sex in their N-termini.10,13 Despite these similarities, SIAH proteins can hormone estrogen (17b-estradiol) induces SIAH2 at the mRNA and have specific substrates (Table 1). Cancer-relevant functions of protein levels in human breast cancer cells.27 Surprisingly, it is still SIAH1 are influenced by SIAH isoforms. For example, the SIAH1 unclear whether these transcription factors control SIAH splice variant SIAH1S inhibits SIAH1 and promotes chemotherapy expression in cells of hematopoietic origin. resistance and breast tumorigenesis.14 SIAH1S has so far not been Due to self-ubiquitinylation, SIAH proteins are very unstable and reported in leukemias. A 49-amino-acid shorter form of SIAH2 often hardly detectable. Homo- and heterodimerization control (B32 kDa, D23–71) has been detected in human bone marrow,12 the steady-state levels of SIAHs, and SIAH2 promotes proteasomal and this protein is a highly active ubiquitin ligase.12,15,16 degradation of SIAH1.10,28 Self-degradation of SIAHs is often a SIAHs act alone or are an essential part of multi-protein ubiquitin- marker for their ability to act as ubiquitin-ligases. The ligase complexes encompassing SIAH1. These can contain the SIAH- deubiquitinylating enzyme USP13,29 and Vitamin K3,30 limit SIAH interacting protein/Calcyclin-binding protein (SIP/CACYBP) and the autodegradation and thereby its activity in kidney-derived cells adapters SKP1 and TBL1/EBI. SIP is suggested to position substrates and melanomas. and E2 enzymes for SIAH1-dependent poly-ubiquitinylation.10,17 Further physiological and pharmacological stimuli can mod- Interestingly, SIP has critical relevance for lymphoid development ulate SIAH functions (Table 2). Genotoxic drugs and hypoxia and cell growth.18,19 A further adapter for SIAH1 is the zinc finger modulate the expression and activities of SIAHs p53 dependently protein plenty of SH3-domains (POSH), which accentuates pro- and independently.11,23,24,31–34 DNA damage triggers an inhibitory apoptotic effects of SIAH1.20 Many direct interaction partners of phosphorylation of SIAH1 at serine-19 that is catalyzed by the SIAHs share the peptide sequence PxAxVxP (VxP, core sequence; kinases ATM/ATR.32,33 The tumor suppressive kinase HIPK2 x, any amino acid). Mutational analyses show that this so called phosphorylates SIAH2 at the N-terminal serines-26,-28,-68. These SIAH ‘degron’ (abbreviation for ‘degradation on’) is a consensus inactivating phosphorylation events stabilize SIAH2 and equally binding motif for SIAHs and hence critical for proteasomal disrupt its interactions with HIPK2, which itself is targeted by degradation.10,14,17,21,22 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 REGULATION OF SIAH EXPRESSION AND ACTIVITY alter their activity toward substrates. For example, the stress- In the murine myeloblastic cell line M1 (generated from a inducible p38 kinase promotes phosphorylation of SIAH2 at spontaneous leukemia), expression of a stably introduced threonine-24 and serine-29 and a phospho-mimic mutant of

Table 1. Examples of leukemia-relevant proteins targeted by SIAHs

Target Hematopoietic phenotype SIAHs experimentally proven Regulators of proteasomal References to induce proteasomal degradation degradation

Leukemia fusion proteins AF4-MLL AML, ALL SIAH1 Taspase1, HDACi 12,52 AML1-ETO AML SIAH1 SIAH degron?, HDACi 35,69 PML-RARa AML (APL) SIAH1/SIAH2 Coiled-coil domain, HDACi 16,35,72

Other proteins FLT3-ITD AML, ALL SIAH1/SIAH2 Tyrosine phosphorylation, 15,76 HDACi ELL2 AML SIAH1 AFF4 13 HDAC3 AML, DLBCL (ABC type), macrophage SIAH2 TBL1, GPS2, TBLR1, SKI 101,106,107,109 development NCOR AML, macrophage development SIAH1/SIAH2 TBL1, GPS2, TBLR1, SKI, 107–110 estrogen BOB.1/OBF.1 B cell development, B cell leukemias SIAH1 SIAH degron 74 VAV1 lymphoid malignancies, hematopoietic SIAH2 Tyrosine phosphorylation 83 differentiation b-catenin AML, thymocyte development SIAH1/SIAH2 SIP/CACYBP, p53, E2F1 17,18,26,111,112 Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; APL, acute promyelocyctic leukemia; CACYBP, Calcyclin-binding protein; DLBCL, diffuse large B cell lymphomas; HDACi, histone deacetylase inhibitor; ?, speculative. AML1 is also known as Runt-related transcription factor-1 (RUNX1) or core-binding factor alpha-2 (CBFA2); BOB.1/OBF.1 is also known as OCA-B, BOB1, OBF-1. AML carrying the translocation t(8;21) is classiﬁed as the French–American–British (FAB) AML-M2 subtype; APL harboring the translocation, t(15;17) represents the FAB M3 subtype; acute monoblastic/monocytic leukemia is the AML-M5 subtype.

& 2013 Macmillan Publishers Limited Leukemia (2013) 792 – 802 Emerging impact of SIAH1 and SIAH2 in leukemia OH Kra¨mer et al 794 Table 2. Factors modulating SIAH expression and function

Agent Effect on SIAHs Cell type References

Anti-IgM Induction of SIAH1 B cell lymphoma 74 glycoprotein CD38 suppression of SIAH1 expression primary B cells DNA damage Inhibition of SIAH1 Solid tumor-derived cells and MEFs 32 DNA damage Inhibition of SIAH1 Solid tumor-derived cells and neurons 33 DNA damage Accumulation of SIAH1 20 17b-estradiol Induction of SIAH2 Breast cancer 27 Hexachlorophene Induction of SIAH1 B cell lymphoma 114 HMBA or prostratin Suppression of SIAH1 expression Cervix carcinoma 13 HDACi Induction of SIAH1 induction of UBCH8a Embryonic kidney derived cells and AML 35 HDACi 15,16,35,117 Hypoxia Induction of SIAH2 Solid tumor-derived cells and MEFs 31 Inhibition of SIAH2 11,34 Metabolic stress Activation of SIAH1 MEFs 19 Vitamin K3 Inhibition of SIAH2 Melanoma 6 Abbreviations: AML, acute myeloid leukemia; HDACi, histone deacetylase inhibitor; HMBA, hexamethylene bisacetamide; MEFs, murine embryonic ﬁbroblasts. aE2 ubiquitin-conjugase for SIAHs.

SIAH2 localizes to perinuclear regions.34 Moreover, the nuclear These data, together with several reports summarized below, export of SIAH2 is sensitive to leptomycin B (which blocks the suggest that SIAH1 functions as a tumor suppressor. Nevertheless, export protein CRM1/exportin-1),34 and it is plausible that such recent evidence argues for a pro-tumorigenic role of SIAHs, and mechanisms are altered in certain cells. For most of the above particularly for SIAH2, in solid tumors. For example, Siah2 defici- mentioned mechanisms further experimental testing is required ency attenuates the formation of aggressive prostate carcinomas to see whether they equally operate in blood cells and whether in mice and SIAH2 expression correlates with lung tumorigen- different chemotherapies induce distinct relevant post- esis.10,11,40 Moreover, outcompeting SIAHs with small peptides translational modifications of SIAHs. reduces breast cancer and melanoma growth and spread.10 Activities of SIAHs are also controlled by the abundance of E2 However, the biological consequences of SIAH inactivation in ubiquitin conjugases. SIAH2 preferentially interacts with the E2 normal tissues of adult animals are unknown. Data from primary UBCH8 and SIAH1 cooperates with UBCH8 and E2s of the UBCH5 tumors show that SIAH1 locates to a region frequently lost in family.13,16,35 Of note, HDACi induce UBCH8 to accelerate the tumors. Allelic loss at 16q12.1 encompassing SIAH1 has, for proteasomal degradation of oncoproteins and to eliminate example, been reported for hepatocellular carcinomas.10,41 leukemic cells.1,15,16,35,36 Moreover, HDACi induce an Inactivating mutations of SIAH1 have so far only been identified accumulation of SIAHs35 (and our unpublished data). Details on in gastric cancers,42 and data on putative deletions or mutations the control of leukemogenic factors by HDACi are summarized in of SIAHs in leukemias are not available yet. the following sections. We will now discuss leukemia-relevant targets of SIAH1 and SIAH2, and how molecular mechanisms involving SIAH-dependent proteasomal degradation operate in leukemic cells. SIAHS AND TUMORIGENESIS Knockout animals can provide valuable insights into protein functions. Mice lacking both Siah1a and Siah2 are not viable, but LEUKEMIA FUSION PROTEINS animals with individual knockouts are available. Siah1a knockout AF4 and AF4/MLL mice are severely growth retarded and often die early, with poor One of the most frequent genetic aberrations of the human mixed bone formation and twice as much osteoclasts as their wild-type lineage leukemia (MLL) gene is the chromosomal translocation counterparts.10 Siah2 null mice appear rather normal, but they t(4;11)(q21;q23) involving ALL1 fused gene on chromosome-4 (AF4; present an expansion of myeloid precursors in the bone marrow four-eleven-leukemia, FEL).43 This abnormality is recurrently found suggesting a myeloproliferative phenotype. This effect particularly in acute leukemia patients and results in AF4/MLL and MLL/AF4 occurs in cells sensitive to macrophage colony-stimulating factor, (derivative 11/derivative 4-leukemia; Figure 1a). In t(4;11) leukemia, a cell type which can differentiate into osteoclasts.37 These one fusion derivate is proteolytically cleaved by the protease findings indicate that SIAHs modulate the immune system and threonine-aspartase-1 (Taspase1). This limited proteolysis stabi- this can critically determine tumorigenesis. Furthermore, they lizes the SIAH-containing complex and traps SIAHs.43–45 support to investigate the physiological relevance of SIAHs in the Genetic irregularities of MLL are critical steps in the malignant hematopoietic system. transformation of hematopoietic precursors to acute myeloid Notably, SIAH1 can form part of a molecular signature for tumor leukemia (AML) or acute lymphoblastic leukemia (ALL). MLL encodes reversion in leukemias and other tumors. In such rare anti- a histone methyltransferase ensuring proper cell cycle transitions malignant programs, cancer cells lose their transformed state, and precise expression of developmentally important homeobox which stems from chromosomal instability, translocations, onco- genes.43,46,47 The ubiquitously expressed multi-domain protein gene addiction and tumor suppressor inactivation.11,23,38 MLL is processed into an N-terminal MLLN (p300) and a C-terminal Expression of the multidrug resistance protein MDR1 is a MLLC fragment (p180) by Taspase1. These form heterodimers via general problem in cancer treatment. Remarkably, SIAH1 internal FRYC/FRYN domains (Figure 1a), to ensure stable steady- reduces transcription of MDR1 by activating JUN kinase and state levels and the correct subnuclear localization of MLL.48 binding of the transcription factor JUN to the MDR1 promoter.39 The resulting mature MLLN320/C180 heterodimers tri-methylate These data demonstrate that SIAHs exert crucial functions apart histone H3 via their C-terminal SET domains to influence gene from the induction of proteasomal degradation. expression,43 and they assemble protein supercomplexes.49

Leukemia (2013) 792 – 802 & 2013 Macmillan Publishers Limited Emerging impact of SIAH1 and SIAH2 in leukemia OH Kra¨mer et al 795

Figure 1. Interplay of MLL, AF4-MLL, SIAH and Taspase1 in t(4;11) leukemias. (a) MLL activation by Taspase1. Autoproteolysis of the Taspase1 proenzyme into the subunits a28 and b22 produces the active enzyme, which subsequently hydrolyzes the MLL protein. This allows inverse heterodimerization of the emerging cleavage products MLLN and MLLC and formation of a stable high molecular-weight protein complex. (b) AF4 as well as its unprocessed fusion allele AF4-MLL bind SIAHs via their ALF domain and thereby become subjected to poly- ubiquitinylation and proteasomal degradation. Cleavage of the AF4-MLL protein by Taspase1 allows heterodimer formation of the resulting subunits into a stable multi-protein complex resistant to SIAH-mediated proteasomal degradation. Thus, a Yin-Yang situation is established by Taspase1 stabilizing AF4 and AF4-MLL and SIAHs contributing to the proteasomal degradation of AF4 and AF4-MLL. Accumulation of those high molecular weight complexes in addition to a supposed trapping of SIAHs in this complex might have a causal role in leukemogenesis. CS: Taspase1 cleavage sites CS1 and CS2 yellow; ALF: AF4/LAF4/FMR2 family; the AF4/MLL breakpoint is indicated. Note that drawings are not to scale, complex binding partners are adumbrated.

Chromosomal translocations of the human MLL gene are a pathological AF4-MLL fusion protein still interacts with SIAHs to hallmark of aggressive pediatric and adult leukemias with rather become subjected to proteasomal degradation. Similar to MLL, poor prognosis.50,51 In the majority of leukemia cells carrying MLL AF4-MLL is processed by Taspase1. Upon cleavage by Taspase1, translocations, wild-type MLL is still expressed. Apparently, the the AF4-MLL.N and MLL.C cleavage products become protected fusion proteins exert dominant disease mechanisms and as such and the complex including SIAHs and AF4-MLL is stabilized. This represent promising targets.43,49 MLL-AF4 was postulated to be molecular mechanism is supposed to trap SIAHs,12 and to the oncogene in t(4;11) leukemogenesis when it was discovered.48 consequently disturb steady-state protein levels relying on SIAH However, 80% of cases with translocations involving AF4 also carry activity against tumor-relevant proteins.32 Chemico-genetic AF4-MLL and nearly all established t(4;11) cell lines express both targeting of Taspase1 would thus not only counteract onco- rearranged alleles.43 Indeed, recent evidence shows that AF4-MLL genic AF4-MLL accumulation but also SIAH trapping and drives cellular transformation and leukemia induction, whereas disturbed proteasomal homeostasis. However, Taspase1 is not MLL-AF4 rather disturbs cell cycle control and apoptosis.52 The affected by available protease inhibitors and no genetic trans- absence of the AF4-MLL allele in some leukemic cells might be dominant-negative mutants are available yet.55,57 Although all explained by the fact that many oncoproteins work based on a consequences of the SIAH/AF4-MLL interaction remain to be ‘hit-and-run mechanism’; AF4-MLL might be required during understood, these data demonstrate a crucial role for SIAHs in disease onset but is lost or even dispensable due to secondary leukemogenesis driven by AF4-MLL. In light of such findings, it is aberrations.12,43 surprising that nothing is known about a putative effect of SIAHs To execute their (patho)biological functions, the 500 kDa on the stability of Taspase1 and vice versa. precursor of MLL as well as its oncogenic MLL-fusions have to Remarkably, a recent report revealed that HDACi evoke cell be processed proteolytically (Figure 1).48,53,54 Taspase1 cleaves death of t(4;11)-positive primary infant acute lymphoblastic substrates in trans by recognizing a conserved peptide motif leukemia cells, and downmodulation of MLL-AF4 on the mRNA 0 0 0 0 (Q3[F,I,L,V]2D1kG1 x2 D3 D4 ).48,54 Following autoproteolysis of the and protein levels was observed in the t(4;11)-positive precursor B Taspase1 zymogen (cis-cleavage), its subunits assemble into an cell acute lymphoblastic leukemia line SEM.58 It is possible that the ab-heterodimer possibly representing the active ‘monomeric’ induction of UBCH8-dependent SIAH functions attenuates AF4- protease (Figure 1a). Mutation of the catalytic nucleophile, MLL and it is likewise interesting to see whether HDACi may affect threonine-234, inactivates Taspase1 in cis and in trans.55 Albeit Taspase1. Extrapolating our current knowledge, one could expect Taspase1 localizes predominantly in the nucleus, its interac- synergistic therapeutic benefits from rationally designed tion with NPM1 allows transient access of the protease to the combination regimen. Applying HDACi with still to be identified cytoplasm.56 MLL processing is expected to occur in the cytoplasm Taspase1 inhibitors should prevent AF4-MLL stabilization and or during nuclear import, when MLL/Taspase1-complexes are consequently accelerate its SIAH-dependent degradation. As rapidly imported into the nucleus via importins.54,56 aberrant recruitment of the methyltransferase hDOT1L to MLL AF4 and AF4-MLL share the first three exons of the AF4 gene. via the AF10 part alters histone H3 methylation at K79 and These exons encode 360 amino acids comprising the N-terminal activates gene expression promoting leukemogenesis, drugs half of the ALF domain including the SIAH-dependent degron blocking DOT1L may also be given with HDACi to correct (Figure 1), which allows an exceptionally tight regulation that aberrant gene expression driven by MLL fusions and DOT1L.59 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 AML1-ETO and PML-RARa SIAHs and exerts oncogenic activity when transported to the The transcription factor acute myeloid leukemia-1 (AML1) and the nucleus.44,45 In the physiological situation, the unprocessed core-binding factor-b (CBFb) form the transcriptional activator CBF.

& 2013 Macmillan Publishers Limited Leukemia (2013) 792 – 802 Emerging impact of SIAH1 and SIAH2 in leukemia OH Kra¨mer et al 796 Aberrations of AML1 are judged as characteristic initiating events BOB.1.74 This B cell-specific coactivator interacts with SIAHs in B in leukemogenesis.60 The AML1-ETO fusion originates from a and T cells and evokes redistribution of SIAH1 to the nuclear balanced translocation involving AML1 and the gene encoding the compartment.74 These observations are also reminiscent of the transcriptional repressor eight-twenty-one (ETO). AML carrying this trapping of SIAHs by AF4-MLL (Figure 1b); AML1-ETO and PML- translocation t(8;21)(q22;q22) accounts for 5–12% of AML cases, RARa may disturb protein degradation by titrating them away which renders it the most common translocation reported in from other cancer-relevant substrates. AMLs.61 In AML1-ETO, nearly the entire open reading frame of ETO It is equally thinkable that wild-type proteins and leukemia fuses with the runt homology domain of AML1, which controls fusion proteins are differentially degraded in the presence of AML1 target genes ensuring proper hematopoietic differentiation. HDACi because they variably depend on the HSP90 chaperone Consequently, AML1-ETO represses differentiation genes by facilitating protein folding and stability. HDACi were reported to recruiting transcriptional repressors, histone deacetylases and induce an inhibitory acetylation of HSP90 to liberate oncogenic associated corepressors, via its ETO portion.5,60 Besides the proteins for proteasomal degradation.75,76 However, HDACi aberrant AML1-ETO-mediated gene repression, AML1-ETO blocks targeting class I HDACs (HDACs1,-2,-3,-8) can reduce leukemia- other transcription factors, such as C/EBPa, c-JUN, PLZF, SMAD3, relevant proteins, although they cannot affect HSP90, which is VDR and p53.62 Curiously, a splice variant of AML1-ETO, AML1- specifically deacetylated by the class IIa deacetylase HDAC6.35,76,77 ETO9a lacking its C-terminal NHR3/NHR4 domains recruiting It is plausible that both HSP90 inhibition and induction of enzymes HDACs and corepressors, has been isolated from human AML of the UPS including UBCH8-SIAH1/-SIAH2 operate in a concerted cells. This protein turned out to be more oncogenic than AML1- manner. ETO itself in murine models.63 Although the above data suggest HDACi as a strategy against Aberrant repression of genes inducing differentiation is also AML1-ETO-positive cells, acetylation of lysine-43 by the acetyl- linked to the development of acute promyelocyctic leukemia. transferase p300 can promote oncogenesis driven by AML1- Acute promyelocyctic leukemia represents 5–8% of all AML cases ETO9a.78 Acetylation-dependent activation of this oncoprotein and is characterized by the PML-RARa protein, which results from though contrasts previous data and the anti-leukemic effects of a fusion between the promyelocytic leukemia (PML) and retinoic HDACi toward AML1-ETO-positive cells.8,35,61,67–70,79 Nonetheless, acid receptor-a (RARa) gene (t(15;17)(q22;q21)). PML-RARa disturbs the HDACi-inducible proteasomal degradation of AML1-ETO may RAR target gene expression promoting maturation of the trump acetylation-dependent effects. One can also speculate that granulocytic lineage. Albeit stimulation with retinoic acid releases acetylation of AML1-ETO and of other proteins is required in corepressors from the RARa/RXR heterodimer, the oncogenic PML- certain tumor stages and dispensable or even damaging in others. RARa recruits corepressor complexes and DNA methylating It is equally possible that HDACi affect p300 in AML1-ETO-positive enzymes that resist physiological levels of retinoic acid.5,64 cells, and hyperacetylation of AML1-ETO in HDACi-treated cells Moreover, PML suppresses tumor growth by facilitating the might trigger its destabilization. acetylation and stabilization of p53 and this property is lost in Another open question is the putative role of p53 for the PML-RARa.5,62,64 Data from murine models suggest that PML-RARa proteasomal degradation of AML1-ETO and PML-RARa. Although requires additional genetic changes to induce aggressive acute it is known that both factors antagonize p53,5,62,64 which can induce promyelocyctic leukemia.65,66 Although it is clear that chromo- SIAH1 and SIAH2 in certain cells,23–25 it is unclear whether leukemia somal translocations found in AML can encode transcription fusion proteins attenuate their proteasomal turnover by inactivating factors disrupting proper hematopoietic transcription, PML-RARa the p53 pathway. Interestingly, AML1-ETO-expressing cells are also is yet the only such oncogenic fusion protein that can be targeted sensitive to corticosteroids, which like HDACi evoke a proteasome- specifically. Acute promyelocyctic leukemia patients have a dependent depletion of AML1-ETO.80 SIAH-dependent proteasomal favorable prognosis as they respond to pharmacological doses degradation could also eliminate the more oncogenic AML1- of retinoic acid-mediating dissociation of corepressor complexes ETO9a63 and treatment-resistant PML-RARa,81 as long as they still from PML-RARa, followed by its proteasomal and caspase- contain SIAH recognition motifs (Figure 2). This might, for dependent degradation.5,16,62 example, be the case for arsenic trioxide-resistant PML-RARa Several reports demonstrate that HDACi kill AML1-ETO-positive mutants harboring mutations in the PML B2 domain preceding cells in vitro,8,35,67–70 and HDACi preferentially induce degradation the coiled coil.81 Such notions support the usage of HDACi to of AML1-ETO and PML-RARa over the intact proteins.35,69,71 The increase the success of chemotherapies and to prevent the advent underlying mechanism might be that AML1-ETO and PML-RARa of leukemic clones with additional aberrations in leukemia fusion are preferred targets for proteasomal degradation induced by proteins. Further leukemia fusion proteins might also be sensitive UBCH8 in conjunction with SIAH1 and SIAH2 (Figure 2a).16,35,72 to the HDACi-induced UBCH8-SIAH module; an example could For PML-RARa and PML, the coiled-coil domain was identified be PAX5-PML containing the PML coiled coil.82 One should as the structural determinant for SIAH-mediated proteasomal though keep in mind that not any leukemia fusion protein is a degradation.72 PML-RARa hence represents an example for target of SIAHs, for example, STAT5-RARa (17q21.1-q21.2) is not,35 a target not dependent on the SIAH degron (Figure 2b). and not all binding partners of SIAHs become degraded by Furthermore, SIAHs supersede the stabilization of PML-RARa by proteasomes.16,83 Such findings illustrate the specificity of the the putative ubiquitin-ligase TRIAD1.16 It is not absolutely clear SIAH-UPS-axis. 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 FLT3 MUTANTS AND FURTHER SIGNALING MOLECULES occurrence of four SIAH recognition motifs in AML1-ETO and FMS-like tyrosine kinase-3 (FLT3), which crucially controls hema- only two in AML1 and ETO suggests that this molecular feature topoiesis, belongs to the type III receptor tyrosine kinases attracts SIAHs to AML1-ETO (Figure 2b). Functionally relevant subfamily, also including c-KIT, c-FMS and PDGF-receptors.84 dimerization and oligomerization of fusion proteins5,61,73 allows FLT3 contributes to the proliferation, differentiation and survival the clustering of SIAH degrons and this may further concentrate of hematopoietic stem cells. In normal bone marrow, FLT3 is SIAHs proximal to AML1-ETO and PML-RARa. Such a mechanism mainly expressed in early CD34-positive myeloid and lymphoid might equally explain why SIAH1 is a nuclear protein in AML1- progenitors and its expression ceases during differentiation.85 ETO- or PML-RARa-positive leukemic cells, albeit SIAH1 occurs in Activating mutations of FLT3 tie in with poor clinical outcome the cytosol or dispersed throughout the cell in cells lacking these and occur in 20–30% of AML patients. The most common FLT3 fusions.15,16,35 Of note, similar findings were made for SIAH1 and mutations in AML are internal tandem duplications (ITDs).86 These

Leukemia (2013) 792 – 802 & 2013 Macmillan Publishers Limited Emerging impact of SIAH1 and SIAH2 in leukemia OH Kra¨mer et al 797

Figure 2. The UBCH8-SIAH-axis in AML1-ETO-, PML-RAR, and FLT3-ITD-positive leukemias. (a) Whereas FLT3 is targeted to the plasma membrane, FLT3-ITD mainly accumulates at the endoplasmic reticulum. Constitutive phosphorylation of FLT3-ITD enhances its binding to SIAH and subsequent poly-ubiquitinylation and proteasomal degradation. The nuclear leukemic fusion proteins AML1-ETO and PML-RAR are also degraded via the SIAH1-UBCH8-axis, which is further enhanced by the HDACi-induced transcriptional activation of the UBCH8 gene. The Yin-Yang is between leukemia fusion proteins contributing to transformation and SIAHs blocking this undesired effect by accelerating poly-ubiquitinylation and proteasomal degradation of leukemogenic proteins. (b) SIAH binding to leukemogenic proteins is conferred via different mechanisms, which can involve UBCH8 as an E2 ubiquitin-conjugase. The occurrence of four SIAH degron recognition motifs (VxP) in the AML1-ETO fusion and only two in AML1 and ETO may attract quantitatively more SIAHs to AML1-ETO and enhance its proteasomal degradation. Whereas PML-RAR binds SIAH via the coiled-coil domain located in the PML part of the leukemic fusion protein, tyrosine phosphorylation of the FLT3-ITD mutant is a prerequisite for SIAH binding. The induction of UBCH8 by HDACi turned out as a limiting factor for the proteasomal degradation of FLT3-ITD in AML cells and in heterologous expression systems. Note that drawings are not to scale; important protein domains, motifs and modiﬁcations, and the proteasomal complex are indicated; Ub, ubiquitin.

locate to the regulatory juxtamembrane region, which regulates FLT3 mutants whether they are resistant to TKi or not. Accordingly, the access of adenosine triphosphate to the kinase domain. Small HDACi act as very potent drugs in combination with TKi.15,76,88 fractions of patients suffering from chronic myeloid leukemia, Interestingly, expression of FLT3 is controlled by factors being myelodysplasia and ALLs also carry FLT3 mutants.85 FLT3-ITDs regulated by SIAHs. For example, the FLT3 promoter is directly show ligand-independent dimerization and cause cytokine- controlled by MLL-AF4 and MLL fusion-positive leukemias express independent receptor activation promoting cell proliferation high levels of FLT3 mRNA.43,92 Thus, MLL-AF4 might accelerate linked to STAT5 activation and malignancy.87,88 FLT3-ITD is leukemogenesis in the context of mutated FLT3. Mutagens and sufficient to induce a myeloproliferative phenotype and in some cooperating lesions such as FLT3-ITD also collaborate with AML1- cases also lymphoid disease in transgenic or bone marrow- ETO and PML-RARa in inducing AML.5,65 Hence, SIAHs seem to transplanted mice.86 have bifurcating antagonistic effects on class I mutations halting As FLT3-ITD is a disease-associated ‘driver’ mutation, it is a valid proper differentiation and class II mutations conferring therapeutic target.86 Drugs targeting constitutively active FLT3 proliferation and survival. Thus, HDACi inducing UBCH8 and undergo extensive clinical testing and AC220 is currently the most SIAHs may be particularly useful against such diseases specific inhibitor for the catalytic activity of FLT3.86,88,89 (Figure 2a).15,35,62 Unfortunately, FLT3-ITD-positive AML patients develop resistance Clearly, it is worth investigating if additional transcription to AC220 under therapy.86,89 FLT3 inhibitor development factors and kinases are controlled by SIAHs and can be therefore aims to target drug-resistant FLT3-ITD mutants.86,90,91 pharmacologically controlled. For example, p95vav/VAV1 (a hema- It appears promising that the clinically used pan-HDACi LBH589 topoietic proto-oncogene activated by IGF/IGF-1R), which is was found to enhance the proteasomal elimination of FLT3-ITD at required for antigen-dependent signaling in lymphocytes and therapeutically relevant concentrations.15 SIAH1 and SIAH2, in subject to control by SIAH2,83 may be sensitive to HDACi. conjunction with the HDACi-inducible UBCH8, catalyze poly- Curiously, in Siah2 À / À mice no regulation of TRAF2, VAV1 and ubiquitinylation and proteasomal degradation of FLT3-ITD OBF.1 was found, while these animals presented an expansion of (Figure 2a).15 This effect of SIAHs toward FLT3-ITD is specific and myeloid progenitors.37 It should be noted that interaction of not evoked by the ubiquitin-ligases HDM2, c-CBL and RLIM.15 SIAH2 with VAV1 does not accelerate its proteasomal degradation Apparently, SIAHs recognize FLT3-ITD dependent on its tyrosine but blocks its signaling to JUN kinase and the transcription factors phosphorylation (Figure 2b), which indicates that they particularly AP1 and NFAT. This mechanism requires the N-terminus of SIAH2 eliminate mutant and not wild-type FLT3 ensuring hematopoiesis. and might be linked to T-cell receptor (TCR) stimulation and Future research will clarify how SIAHs, which have no known motif interleukin-2 expression.83 Further interesting candidate for binding phosphorylated proteins, recognize tyrosine phos- molecules that are putatively affected by HDACi comprise the phorylated FLT3 and whether this is valid for all FLT3 mutants. It BCL2-associated athano-gene-1 (BAG1) and the transmembrane may be that this ability of SIAHs is conferred by interaction receptor DCC. Both molecules are targets of SIAHs.10,11 BAG1 is partners within SIAH complexes.10,14 Independent of these details, required to sustain expression of the anti-apoptotic factors BCL2, HDACi-inducible UBCH8-SIAH-axes should target phosphorylated BCL-XL, MCL1 in human AML cell lines,93 and DCC is a prognostic

& 2013 Macmillan Publishers Limited Leukemia (2013) 792 – 802 Emerging impact of SIAH1 and SIAH2 in leukemia OH Kra¨mer et al 798 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 Figure 3. Complex regulatory circuits control SIAH2 and its target disorder.99 The proteasomal inhibitor bortezomib is used against proteins. Chromosome 7q encodes for the micro RNA miR-29a that this incurable disease,7 and SIAH-dependent proteolysis may suppresses SKI expression via its 30-untranslated region (30-UTR). affect multiple myeloma development. Accordingly, loss of chromosome 7q enhances expression of the The pan-HDACi AR-42 is currently in phase II clinical trials for the leukemia-relevant transcriptional co-repressor SKI, which blocks the treatment of relapsed or recurrent hematological and solid ubiquitin-ligase SIAH2 and its activity against HDAC3 and NCOR. SKI cancers. It was recently found that this agent shuts down might indirectly repress the lineage-specific transcription factors PU.1 and RAR, and SKI might promote cellular resistance to stress via constitutively active mutants of KIT in spontaneously developed 77 indirect STAT3 activation. Such aberrations result in a pathological primary canine mast cell tumors. Thus, KIT might be controlled alteration of degradation patterns and an aberrant suppression of via the UBCH8-SIAH-axis. Because AML1-ETO increases expression hematopoietic differentiation. Regarding the Yin-Yang, which one of KIT and KIT mutations are frequently found in CBF-AML patients may call ‘Yin-SIAHng’ of leukemogenesis, SIAHs antagonize aberrant (including t(8;21) and inv(16));100 HDACi could attack such transcriptional control by accelerating the proteasomal elimination leukemic cells via both, targeting Kit as well as AML1-ETO. of (co)repressors.

TRANSCRIPTION AND ELONGATION FACTORS monocytic leukemia (5–10% of adult AMLs). In turn, activation of Data on leukemia fusion proteins show that aberrant transcrip- FLT3 triggers loss of NCOR, and its ablation potentiates FLT3 tional repression involving HDACs is associated with cancer. The ligand-induced proliferation. Thus, therapeutic restoration of NCOR leukemia-relevant transcriptional co-repressor SKI represses reti- may be a therapeutic strategy.108 However, caution is required noic acid signaling by stabilizing HDAC3. SKI binds HDAC3 and as differentiation genes are suppressed by NCOR/SMRT/HDAC- SIAH2 to block the ubiquitin-ligase activity of SIAH2.101 complexes associating with leukemia fusion proteins.8 As NCOR Remarkably, SKI can act as an oncoprotein via its interaction tightly interacts with HDAC3 and both are controlled by SKI and with the lineage-specific transcription factor PU.1. PU.1-dependent SIAH in adherent cells,107,109 SIAHs might relieve aberrant trans- transcription stimulates the terminal differentiation of myeloid criptional repression exerted by leukemic fusions (Figures 2a and 3). cells into macrophages, and loss of PU.1 is linked to myeloid Several additional molecules control NCOR/HDAC3-dependent leukemia.102 As SKI represses PU.1 by recruiting HDAC3, transcriptional control. TBL1, its homologs GPS2 and TBLR1 stabilization of HDAC3 at differentiation-inducing promoters by interact with HDAC3, NCOR and SMRT.107 This complex mediates blocking SIAH2101 may equally contribute to leukemogenesis corepressor/coactivator exchange reactions by a ligand- (Figure 3). Interestingly, the SKI-dependent SIAH2-HDAC3 module dependent recruitment of the 19S proteasome.107 In may be controlled by non-coding microRNAs (miRs) in AML with macrophages, NCOR controls c-JUN/c-FOS-dependent networks aberrant chromosome-7 (-7/del7q). These show upregulation of regulating inflammation, migration and collagen catabolism. SKI and loss of miR-29a on chromosome 7q.103 Further analyses Phosphorylation of c-JUN induces removal of NCOR/HDAC3/ are needed to judge whether miR-29a modulates SKI and SIAH2 to TBL1/TBLR1 complexes by ubiquitinylation complexes containing antagonize the development of this highly aggressive AML. SIAH2.110 Therefore, it is plausible that SIAH2-dependent HDAC3 is also relevant for the pathogenesis of DLBCLs (diffuse proteolysis is critical for myeloid differentiation and inflammation. large B cell lymphomas), which can be divided into three BOB.1/OBF.1 is a coactivator for OCT transcription factors and subtypes. DLBCLs arrested during plasmacytic differentiation have critical for B cell development and germinal center formation.74 activated gene expression profiles and form the ABC subgroup. IgM-crosslinking of the human germinal center-like Burkitt’s These tumors originate from post-germinal center B cells and are lymphoma line Ramos increases SIAH1 and BOB.1 mRNA more frequently therapy resistant than germinal center B cell expression, but decreases the levels of BOB.1 (Table 2). Further- lymphomas.104 Curiously, ABC lymphomas have higher HDAC3 more, in primary tonsilar B cells expression of the activation levels, constitutive phosphorylation of the cancer-relevant marker CD38 correlates with low SIAH1 mRNA levels.74 From these transcription factor STAT3 and expression of its anti-apoptotic data it was proposed that rapidly dividing germinal center target MCL1. Although STAT3 activation increases chemotherapy centroblasts lose B cell receptor expression and BOB.1 resistance,105 it also confers sensitivity of ABC tumors to the HDACi accumulates. It is unclear whether the interaction of SIAH2 with LBH589.106 It will be interesting to see whether upregulation of BOB.1 affects its stability and whether SIAHs antagonize B cell HDAC3 in certain DLBCLs associates with dysregulation of SIAHs. proliferation via BOB.1. The transcriptional corepressors NCOR and SMRT control The transcription factor b-catenin is a target of SIAH1 and definitive erythropoiesis and myeloid lineage commitment.107 SIAH2,17–19,111 and SIP1 is involved in the p53-inducible SIAH1- FLT3 is negatively regulated by NCOR in acute monoblastic/ mediated degradation of b-catenin.112 Of note, SIP1 À / À

Leukemia (2013) 792 – 802 & 2013 Macmillan Publishers Limited Emerging impact of SIAH1 and SIAH2 in leukemia OH Kra¨mer et al 799 thymocytes fail to fully engage TCR-b gene rearrangements and Future studies will likely reveal that additional proteins are have increased apoptosis rates resulting in reduced thymic controlled by UBCH8-SIAH-dependent proteasomal pathways and cellularity.18 Furthermore, b-catenin signaling is required for hence susceptible to HDACi-induced proteasomal degradation. stemness and the oncogene-induced transformation of Candidates might be the transcription factors LMO2 and WT1. progenitor cells (for example, via MLL-AF9),113 and the E2F1- Overexpression of LMO2 is linked to T-ALL and undergoes dependent induction of SIAH1 suppresses b-catenin signaling.26 proteasomal degradation by a yet unidentified pathway.116 WT1 In B cell lymphomas carrying Epstein-Bar-virus, hexachlorophene is a pan-leukemic marker that is controlled by the HDACi- (Table 2) antagonizes repression of SIAH1 by the viral latent dependent induction of UBCH8 and by the serine protease membrane protein-1.33,114 SIAH1 induced by this chemical is HTRA2/OMI,117,118 and these processes may involve SIAHs. active against b-catenin, and consequently expression of its target Furthermore, HDACi alter immune and inflammatory processes, genes cyclin D1 and c-MYC is reduced and lymphoma growth is for example, via the forkhead transcription factor FOXP3, which halted p53-independently.114 controls regulatory T cell subsets.119 It still always has to be noted Interestingly, SIAH1 and SIP1 are also linked to metabolic that not each protein interacting with SIAHs becomes degraded changes. Both are involved in the proteasome-dependent by the proteasome and that SIAHs can exert functions beyond the attenuation of cytoplasmic amounts of the cyclin-dependent induction of ubiquitin-dependent proteolysis.13,16,39,74,83 kinase inhibitor p27 upon glucose deprivation. In addition, compared with wild-type cells SIP À / À embryonic fibroblasts have augmented cytosolic p27 linked to increased motility and CONCLUDING REMARKS invasiveness.19 Like SIAH1, SIAH2 can affect the cellular Modulation of cancer-relevant signaling by small molecules is metabolism. SIAH2 promotes proteasomal degradation of highly desired and still accepted as the ‘gold standard’ in the cytosolic levels of the dihydrolipoamide S-succinyltransferase, clinics. Recent (pre)clinical research revealed novel opportunities which is a subunit of the mitochondrial a-ketoglutarate dehydro- to directly and indirectly modulate proteasomal networks. Current genase complex catalyzing the conversion of a-ketoglutarate research aims to identify which ubiquitin-ligases target specific to succinyl-CoA.115 This rate-limiting step in the ‘Krebs’/TCA/ substrates for poly-ubiquitinylation and proteasomal degradation, citric-acid cycle allows the generation of energy equivalents and and how these enzymes can be controlled. intermediate molecules. Cancer cells rather produce energy by From the data available we hypothesize that SIAHs are glycolysis than via mitochondrial oxidation (‘Warburg’ effect). druggable enzymes that are relevant for cancer development Super elongation complexes (SECs) promote transcriptional and during therapy. Although the SIAH substrate binding domain elongation stimulating normal and leukemia-associated gene may be a useful structure to block these enzymes,11 SIAHs can expression. Together with MLL, SECs instruct homeotic gene eliminate leukemia fusion proteins, aberrant kinases and expression for pattern formation and development.46 Accordingly, chemotherapy resistance proteins.1,12,13,15,16,35,39,71,72,107 All these several more or less frequent translocation partners of MLL coexist factors are crucial for leukemogenesis and therapy, and this in SECs, which contain the transcription elongation factors P-TEFb, supports the view that SIAHs may antagonize leukemogenesis. ELL1/ELL2, and the scaffold proteins AFF4 and AF4/AFF1.46 ELL2 is Enhanced SIAH activities can be achieved by regulating the a stoichiometrically limiting protein of SECs and is specifically abundance of an ubiquitin-conjugase as in the case of HDACi targeted for ubiquitin-mediated degradation by SIAH1, but not by treatment.1,15,16,35,36 Moreover, the tremendous potential of SIAH2.13 Depending on the dose of SIAH1, the scaffolding factor exploiting aberrations in the UPS as an Achilles heel to attack AFF4 has the ability to protect ELL2 by attenuating the access of tumor cells may set the avenue for developing novel combination SIAH1. AFF4 can though also be subjected to proteasomal therapies.15,75,76,88,96,120 degradation induced by SIAH1.13 This mechanism illustrates the However, caution is equally required, as particularly SIAH2 complex regulatory patterns that govern whether SIAHs can appears to promote cell growth, metastasis and the hypoxic drive degrade a protein. This SIAH1-dependent control of transcriptional of solid tumors.10,11,30–32 Additional data including the analysis of elongation processes can be influenced by small molecules. primary blood cancers are necessary to further understand the The plant-derived phorbol and protein kinase C activator role(s) of SIAHs for leukemogenesis. The next goal is then to prostratin (from the mamala tree bark) as well as the hybrid analyze the biological and functional consequences of the SIAH- polar compound hexamethylene bisacetamide (HMBA; Table 2) substrate relationship and how it can be exploited for enhance accumulation of the transcription elongation factor pharmacological control and the benefit of patients. eleven-nineteen lysine-rich leukemia (ELL2) and thereby formation As summarized in this review, SIAH ubiquitin-ligases represent of SECs. Mechanistically, prostratin and hexamethylene bisaceta- master regulators destroying leukemogenic proteins via intricate mide decrease expression of SIAH1 mRNA and protein levels and protease and kinase signaling pathways. We speculate that reduce ELL2 poly-ubiquitinylation and proteasomal degradation. SIAHs, likely in conjunction with UBCH8, are scavengers that This pathway is discussed as a possible mechanism to promote accelerate the proteasomally mediated elimination of rogue HIV transcription for the elimination of latent viral reservoirs.13 leukemia-relevant proteins.1,8 The basal and induced levels of Interestingly, sequence variations in the 36 amino acid RING UBCH8 and SIAHs may predict responsiveness and therapy domains of SIAH1 and SIAH2 are responsible for their different success for cases in which a leukemogenic driver is sensitive to abilities to induce degradation of ELL2. Of three non-conserved HDACi and UBCH8-SIAH-dependent proteasomal degradation. The exchanges SIAH2 alanine-96 versus SIAH1 serine-88 and SIAH2 loss of oncoproteins may likewise be a valid pharmacodynamic glutamine-106 versus SIAH1 proline-98 appear critical for their marker.62 variable effects toward ELL2.13 These differences do not affect the Collectively, increasing evidence supports a central role of SIAH interaction of SIAH2 with ELL2, but its ability to induce ELL2 proteins for cancer biology. Understanding and exploiting the degradation. Nonetheless, a lack of cofactors for SIAH2 in HeLa functional impact of SIAHs in leukemia, in liaison with newly cervix carcinoma cells might also be the reason for the failure of designed and increasingly specific drugs, may pave the avenue for SIAH2 to induce degradation of ELL2.13 As SIAH2 preferentially new rational therapeutic concepts to fight malignancies. interacts with UBCH8,13 and as HDACi induce UBCH8,8,16,36 these agents may render SIAH2 active against ELL2 to modulate oncogenic transcription. Dimerization of SIAHs and the SIAH2- CONFLICT OF INTEREST 10,28 dependent degradation of SIAH1 could further complicate this Gesine Bug has received honoraria and travel grants from Novartis Pharma GmbH scenario. and from Celgene GmbH. The remaining authors declare no conflict of interest.

& 2013 Macmillan Publishers Limited Leukemia (2013) 792 – 802 Emerging impact of SIAH1 and SIAH2 in leukemia OH Kra¨mer et al 800 ACKNOWLEDGEMENTS 22 Wu H, Lin Y, Shi Y, Qian W, Tian Z, Yu Y et al. SIAH-1 interacts with mammalian We wish to thank all members of our groups and Professor T Heinzel for helpful polyhomeotic homologues HPH2 and affects its stability via the ubiquitin-pro- discussions and fruitful collaboration. Our research is supported by grants from the teasome pathway. Biochem Biophys Res Commun 2010; 397: 391–396. Deutsche Krebshilfe, the Deutsche Forschungsgemeinschaft, and the Wilhelm- 23 Telerman A, Amson R. The molecular programme of tumour reversion: the steps Sander-Stiftung. We apologize for works not cited due to space limitations or an beyond malignant transformation. Nat Rev Cancer 2009; 9: 206–216. oversight on our part. 24 Chan P, Mo¨ller A, Liu MC, Sceneay JE, Wong CS, Waddell N et al. 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