Terapias dirigidas a dianas moleculares: perspectiva actual en la leucemia promielocítica / Molecular targeted therapies: current perspectives in acute promyelocytic leukemia

Introducción: La leucemia promielocítica presenta particularidades biológicas y clínicas con respecto al resto de las leucemias mieloides agudas. El descubrimiento de los detalles moleculares de su patogénesis, posibilitó que su tratamiento, constituya una de las mejores representaciones de la investigación traslacional y esto hace que establezca un modelo para el desarrollo de terapias dirigidas a dianas moleculares con enfoque curativo en pacientes con cáncer. Objetivo: Abordar los principales avances en la terapia de la LPM desde el descubrimiento de los agentes diferenciadores hasta su estado actual. Métodos: Se realizó una búsqueda exhaustiva en bases de datos como Scielo, Pubmed, ScienceDirect, Redalyc y se utilizaron como referencias los artículos actualizados publicados principalmente en los últimos cinco años. Análisis y síntesis de la información: Se abordaron los principales avances en la terapia de este tipo de leucemia, desde el descubrimiento de los agentes diferenciadores hasta su estado actual, haciendo énfasis en su mecanismo de acción y nuevas opciones terapéuticas. Conclusiones: Los aportes realizados en el estudio etiopatogénico y molecular de la leucemia promielocítica y su impacto objetivo en la investigación clínica, constituyen uno de los mejores ejemplos de tratamiento dirigido a alteraciones moleculares específicas y representa un modelo de integración biológica, clínica y terapéutica en beneficio de los pacientes afectados con esta enfermedad(AU)

Introduction: Acute promyelocytic leukemia is a biologically and clinically different type from other acute myeloid leukemias. The discovery of molecular details in its pathogenesis enabled its treatment to constitute one of the best examples of translational research and makes a model for the development of targeted therapies with a curative approach in cancer patients. Objective: To analize the main advances in PML therapy from the discovery of differentiating agents to their current state. Methods: An exhaustive search was carried out in the databases as Scielo, Pubmed, ScienceDirect, Redalyc, and updated articles published mainly in the last five years were used as references. Analysis and synthesis of the information: The article addressed the main advances in the therapy of this type of leukemia, from the discovery of differentiating agents to its current state, emphasizing its mechanism of action and new therapeutic options. Conclusions: The contributions made in the etiopathogenic and molecular study of promyelocytic leukemia and its objective impact on clinical research constitute one of the best examples of treatment aimed at specific molecular alterations and represents a model of biological, clinical and therapeutic integration in benefit of patients affected with this disease(AU)

Follow-up and outcome of the twelve-year experience in adult patients with acute promyelocytic leukemia

ABSTRACT Acute promyelocytic leukemia is a subtype of acute myeloid leukemia, characterized by the presence of neoplastic promyelocytes, due to the reciprocal balanced translocation between chromosomes 15 and 17. Currently, with the use of agents that act directly on this molecular change, such as all-trans retinoic acid and arsenic trioxide, APL has shifted from a highly mortal to a curable disease. However, some cases are still at high risk of death, especially early death, and acquiring a better understanding of the clinical and biological factors involving APL is needed to correctly identify and treat such cases. The early suspected diagnosis and prompt initiation of the target therapy are important for better response rates. The follow-up and outcomes, using real-life data from 44 consecutive APL patients, were studied between 2001 and 2013. The overall survival rate was 82.7% and early death was 16%. Almost all patient deaths were due to severe bleeding, which was confirmed by multivariate analysis, as the most important prognostic factor leading to death. A better understanding the pathogenesis of the hemorrhagic complications in APL is needed, as well as the risk factors associated with early death in APL patients, as this has become synonymous with overall mortality.

When SUMO met splicing.

Spliceosomal proteins have been revealed as SUMO conjugation targets. Moreover, we have reported that many of these are in a SUMO-conjugated form when bound to a pre-mRNA substrate during a splicing reaction. We demonstrated that SUMOylation of Prp3 (PRPF3), a component of the U4/U6 di-snRNP, is required for U4/U6•U5 tri-snRNP formation and/or recruitment to active spliceosomes. Expanding upon our previous results, we have shown that the splicing factor SRSF1 stimulates SUMO conjugation to several spliceosomal proteins. Given the relevance of the splicing process, as well as the complex and dynamic nature of its governing machinery, the spliceosome, the molecular mechanisms that modulate its function represent an attractive topic of research. We posit that SUMO conjugation could represent a way of modulating spliceosome assembly and thus, splicing efficiency. How cycles of SUMOylation/de-SUMOylation of spliceosomal proteins become integrated throughout the highly choreographed spliceosomal cycle awaits further investigation.

Human Regulatory Protein Ki-1/57 Is a Target of SUMOylation and Affects PML Nuclear Body Formation.

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin's lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria.

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions Between SUMO Traps and Mono- or PolySUMO Moieties.

Isolating endogenous SUMOylated proteins is a challenging task due to the high reversibility of this posttranslational modification. We have shown that SUMO traps are useful tools for the enrichment and isolation of proteins modified by SUMO in vitro and in vivo. To characterize the affinity and specificity of different SUMO chains for these traps, that are based on SUMO-interacting motifs, we have used real-time surface plasmon resonance (SPR), which allows a label-free analysis of protein/protein interactions. Here, a protocol to determine the affinities of multivalent SUMO traps for polySUMO chains or mono-SUMO molecules by SPR is presented.

In silico structural and functional characterization of the RSUME splice variants.

RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.

Evaluation of the expression of p53, MDM2, and SUMO-1 in oral lichen planus.

OBJECTIVE: The objective of this study was to compare the expression of proteins p53, MDM2, and SUMO-1 in oral lichen planus (OLP) lesions, epithelial dysplasia, and squamous cell carcinoma. MATERIALS AND METHODS: The sample consisted of the following five groups of cheek mucosa lesions: normal mucosa (NM), inflammatory fibrous hyperplasia (IFH), lichen planus, epithelial dysplasia, and squamous cell carcinoma. The tissue samples were stained with hematoxylin-eosin and submitted to immunohistochemistry using anti-p53, anti-MDM2, and anti-SUMO-1 antibodies. RESULTS: The results of this study demonstrated similar expression of p53 and MDM2 between OLP, oral epithelial dysplasia and, to a lesser extent, between OLP and oral squamous cell carcinoma (OSCC). However, for SUMO-1 a similar expression was observed in OLP, NM, and IFH. CONCLUSIONS: The results demonstrated overexpression of important proteins (p53 and MDM2) related to regulatory mechanisms of apoptosis in OLP, suggesting that there is a favorable environment for malignant transformation. The expression of SUMO-1 in OLP was similar to NM and IFH, suggesting that alterations of this protein occur at later stages of carcinogenesis, because important overexpression occurred in oral epithelial dysplasia and OSCC.

DNA damage-induced heterogeneous nuclear ribonucleoprotein K sumoylation regulates p53 transcriptional activation.

Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a nucleocytoplasmic shuttling protein that is a key player in the p53-triggered DNA damage response, acting as a cofactor for p53 in response to DNA damage. hnRNP K is a substrate of the ubiquitin E3 ligase MDM2 and, upon DNA damage, is de-ubiquitylated. In sharp contrast with the role and consequences of the other post-translational modifications, nothing is known about the role of SUMO conjugation to hnRNP K in p53 transcriptional co-activation. In the present work, we show that hnRNP K is modified by SUMO in lysine 422 within its KH3 domain, and sumoylation is regulated by the E3 ligase Pc2/CBX4. Most interestingly, DNA damage stimulates hnRNP K sumoylation through Pc2 E3 activity, and this modification is required for p53 transcriptional activation. Abrogation of hnRNP K sumoylation leads to an aberrant regulation of the p53 target gene p21. Our findings link the DNA damage-induced Pc2 activation to the p53 transcriptional co-activation through hnRNP K sumoylation.

Human stanniocalcin-1 interacts with nuclear and cytoplasmic proteins and acts as a SUMO E3 ligase.

Human stanniocalcin-1 (STC1) is a glycoprotein that has been implicated in different physiological process, including angiogenesis, apoptosis and carcinogenesis. Here we identified STC1 as a putative molecular marker for the leukemic bone marrow microenvironment and identified new interacting protein partners for STC1. Seven selected interactions retrieved from yeast two-hybrid screens were confirmed by GST-pull down assays in vitro. The N-terminal region was mapped to be the region that mediates the interaction with cytoplasmic, mitochondrial and nuclear proteins. STC1 interacts with SUMO-1 and several proteins that have been shown to be SUMOylated and localized to SUMOylation related nuclear bodies. Although STC1 interacts with SUMO-1 and has a high theoretical prediction score for a SUMOylation site, endogenous co-immunoprecipitation and in vitro SUMOylation assays with the purified recombinant protein could not detect STC1 SUMOylation. However, when we tested STC1 for SUMO E3 ligase activity, we found in an in vitro assay, that it significantly increases the SUMOylation of two other proteins. Confocal microscopic subcellular localization studies using both transfected cells and specific antibodies for endogenous STC1 revealed a cytoplasmic and nuclear deposition, the latter in the form of some specific dot-like substructure resembling SUMOylation related nuclear bodies. Together, these findings suggest a new role for STC1 in SUMOylation pathways, in nuclear bodies.

Avaliação da expressão das proteínas p53, mdm2 e sumo-1 em líquen plano bucal, displasia epitelial bucal e carcinoma de células escamosas bucal / Evaluation of the imunnohistochemical expression of p53,MDM2, and SUMO-1 in oral lichen planus, oral epithelial dysplasia and oral squamous cell carcinoma

O líquen plano (LP) é caracterizado como uma doença crônica de cunho autoimune que apresenta prevalência estimada de 2% na população emgeral. Há grande controvérsia quanto à classificação da Organização Mundial de Saúde de que este seria uma desordem potencialmente maligna. O presente trabalho tem como proposição avaliar a expressão das proteínas p53, MDM2 e SUMO-1 em lesões de LP bucal e comparar a expressão destas proteínas com a observada em displasia epitelial (DE) bucal e carcinoma de células escamosas (CCE) bucal. A amostra por interesse foi constituída por cinco grupos de lesões em mucosa jugal. O primeiro grupo foi constituído por amostras de mucosa com aspecto clínico de normalidade (MN), o segundo por hiperplasia fibrosa inflamatória (HFI), o terceiro por LP, o quarto por DE e o quinto grupo por CCE. Estas amostras teciduais foram submetidas a exame histoquímico pela técnica da hematoxilina-eosina e exame imunoistoquímico para anticorpo anti-p53, anti-MDM2 e anti-SUMO-1. Os dados foram tabulados e tratados pelos testes de Kolmogorov-Smirnov e exato de Fisher. Os resultados do presente estudo mostraram que não houve diferença estatisticamente significante da expressão de p53 quando comparado LP com DE (p=0.2042) e com CCE (p=0.0656), esta diferença estava presente quando o LP foi comparado a HFI (p=0.00001) e a MN (p=0.0007). A diferença na expressão da MDM2 não foi estatisticamente significante quando comparado LP com DE (p=1.0) e com CCE (p=0.9972), mas não estava presente quando o LP foi comparado a HFI (p=0.0005) e a MN (p=0.0052). Em relação à proteína SUMO-1, houve diferença estatisticamente significante quando comparado LP com DE(p=0.0492) e com CCE (p=0.0001), na comparação de LP com HFI (p=1.0) e com MN (p=0.8302) não houve diferença estatisticamente significante...

Lichen planus (LP) is a chronic auto immune disease with an estimated prevalence of 2% in the general population. Controversy exists regarding the World Health Organization classification of LP as a potentially malignant disease. The objective of this study was to investigate the expression of proteins p53, MDM2 and SUMO-1 in oral LP lesions and to compare the expression of these proteins between LP and oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). The sample consisted of the following five groups of cheek mucosa lesions was studied: normal oral mucosa (NM), inflammatory fibrous hyperplasia (IFH),LP, OED, and OSCC. The tissue samples were stained with hematoxy lineosin for histochemical analysis and submitted to immunohistochemistry using anti-p53, anti-MDM2 and anti-SUMO-1 antibodies. The results were analyzed by the Kolmogorov-Smirnov test and Fisher’s exact test. No significant difference in the expression of p53 was observed between LP and OED (p=0.2042) or OSCC (p=0.0656). However, there was a significant difference when LP lesions were compared to IFH (p=0.00001) and NM (p=0.0007). Expression of MDM2 differed significantly between LP and IFH (p=0.0005) and NM (p=0.0052), but not between LP and OED (p=1.0) or OSCC (p=0.9972). A significant difference in the expression of SUMO-1 was observed between LP lesions and OED (p=0.0492) and OSCC (p=0.0001), but not between LP and IFH (p=1.0) or NM (p=0.8302). Further studies are needed to determine the role of inflammation as a possible malignant transformation in cases of LP.

The serine/arginine-rich protein SF2/ASF regulates protein sumoylation.

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

Coxsackievirus B5 induced apoptosis of HeLa cells: effects on p53 and SUMO.

Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin-proteasome system, associated with nuclear aggregation of p53. Striking redistribution of both SUMO and UBC9 was noted at 4 h post-infection, simultaneously with a reduction in the levels of the ubiquitin-ligase HDM2. Taken together, these results suggest that CVB5 infection of HeLa cells elicit the intrinsic pathway of apoptosis by MDM2 degradation and p53 activation, destabilizing protein sumoylation, by a mechanism that is dependent on a functional ubiquitin-proteasome system.

Allelism of Saccharomyces cerevisiae gene PSO10, involved in error-prone repair of psoralen-induced DNA damage, with SUMO ligase-encoding MMS21.

In order to extend the understanding of the genetical and biochemical basis of photo-activated psoralen-induced DNA repair in the yeast Saccharomyces cerevisiae we have identified and cloned 10 pso mutants. Here, we describe the phenotypic characterization and molecular cloning of the pso10-1 mutant which is highly sensitive to photoactivated psoralens, UV(254) (nm) radiation and the alkylating agent methylmethane sulphonate. The pso10-1 mutant allele also confers a block in the mutagenic response to photoactivated psoralens and UV(254) (nm) radiation, and homoallelic diploids do not sporulate. Molecular cloning using a yeast genomic library, sequence analysis and genetic complementation experiments proved pso10-1 to be a mutant allele of gene MMS21 that encodes a SUMO ligase involved in the sumoylation of several DNA repair proteins. The ORF of pso10-1 contains a single nucleotide C-->T transition at position 758, which leads to a change in amino acid sequence from serine to phenylalanine [S253F]. Pso10-1p defines a leaky mutant phenotype of the essential MMS21 gene, and as member of the Smc5-Smc6 complex, still has some essential functions that allow survival of the mutant. DNA repair via translesion synthesis is severely impaired as the pso10-1 mutant allele confers severely blocked induced forward and reverse mutagenesis and shows epistatic interaction with a rev3Delta mutant allele. By identifying the allelism of PSO10 and MMS21 we demonstrate the need of a fully functional Smc5-Smc6 complex for a WT-like adequate repair of photoactivated psoralen-induced DNA damage in yeast.

RSUME, a small RWD-containing protein, enhances SUMO conjugation and stabilizes HIF-1alpha during hypoxia.

SUMO conjugation to proteins is involved in the regulation of diverse cellular functions. We have identified a protein, RWD-containing sumoylation enhancer (RSUME), that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkB in vitro and in cultured cells, leading to an inhibition of NF-kB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1alpha, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells.

PIASgamma represses the transcriptional activation induced by the nuclear receptor Nurr1.

Nurr1 is a transcription factor essential for the development of ventral dopaminergic neurons. In search for regulatory mechanisms of Nurr1 function, we identified the SUMO (small ubiquitin-like modifier)-E3 ubiquitin-protein isopeptide ligase, PIASgamma, as an interaction partner of Nurr1. Overexpressed PIASgamma and Nurr1 co-localize in the nuclei of transfected cells, and their interaction is demonstrated through co-immunoprecipitation and glutathione S-transferase pulldown assays. Co-expression of PIASgamma with Nurr1 results in a potent repression of Nurr1-dependent transcriptional activation of an artificial NGFI-B response element (NBRE) reporter as well as of a reporter driven by the native tyrosine hydroxylase promoter. We identified two consensus sumoylation sites in Nurr1. The substitution of lysine 91 by arginine in one SUMO site enhanced the transcriptional activity of Nurr1, whereas the substitution of lysine 577 by arginine in the second SUMO site decreased transcriptional activity of Nurr1. Interestingly, PIASgamma-induced repression of Nurr1 activity does not require the two sumoylation sites, because each mutant is repressed as efficiently as the wild type Nurr1. In addition, the mutations do not alter Nurr1 nuclear localization. Finally, we provide evidence that Nurr1 and PIASgamma co-exist in several nuclei of the rodent central nervous system by demonstrating the co-expression of Nurr1 protein and PIASgamma mRNA in the same cells. In conclusion, our studies identified PIASgamma as a transcriptional co-regulator of Nurr1 and suggest that this interaction may have a physiological role in regulating the expression of Nurr1 target genes.