A novel mutation in NDUFB11 unveils a new clinical phenotype associated with lactic acidosis and sideroblastic anemia.

NDUFB11, a component of mitochondrial complex I, is a relatively small integral membrane protein, belonging to the "supernumerary" group of subunits, but proved to be absolutely essential for the assembly of an active complex I. Mutations in the X-linked nuclear-encoded NDUFB11 gene have recently been discovered in association with two distinct phenotypes, i.e. microphthalmia with linear skin defects and histiocytoid cardiomyopathy. We report on a male with complex I deficiency, caused by a de novo mutation in NDUFB11 and displaying early-onset sideroblastic anemia as the unique feature. This is the third report that describes a mutation in NDUFB11, but all are associated with a different phenotype. Our results further expand the molecular spectrum and associated clinical phenotype of NDUFB11 defects.

A subcellular analysis of genetic modulation of PINK1 on mitochondrial alterations, autophagy and cell death.

Mutations in the PTEN-induced putative kinase1 (PINK1) represent the second most frequent cause of autosomal recessive Parkinson's disease. The PINK1 protein mainly localizes to mitochondria and interacts with a variety of proteins, including the pro-autophagy protein beclin1 and the ubiquitin-ligase parkin. Upon stress conditions, PINK1 is known to recruit parkin at the surface of dysfunctional mitochondria and to activate the mitophagy cascade. Aim of this study was to use a simple and highly reproducible catecholamine cell model and transmission electron microscopy to characterize whether PINK1 could affect mitochondrial homeostasis, the recruitment of specific proteins at mitochondria, mitophagy and apoptosis. Samples were analyzed both in baseline conditions and following treatment with methamphetamine (METH), a neurotoxic compound which strongly activates autophagy and produces mitochondrial damage. Our data provide robust sub-cellular evidence that the modulation of PINK1 levels dramatically affects the morphology and number of mitochondria and the amount of cell death. In particular, especially upon METH exposure, PINK1 is able to increase the total number of mitochondria, concurrently recruit beclin1, parkin and ubiquitin and enhance the clearance of damaged mitochondria. In the absence of functional PINK1 and upon autophagy stress, we observe a failure of the autophagy system at large, with marked accumulation of dysfunctional mitochondria and dramatic increase of apoptotic cell death. These findings highlight the strong neuroprotective role of PINK1 as a key protein in the surveillance and regulation of mitochondrial homeostasis.

Aberrant recruitment of the nuclear receptor corepressor-histone deacetylase complex by the acute myeloid leukemia fusion partner ETO.

Nuclear receptor corepressor (CoR)-histone deacetylase (HDAC) complex recruitment is indispensable for the biological activities of the retinoic acid receptor fusion proteins of acute promyelocytic leukemias. We report here that ETO (eight-twenty-one or MTG8), which is fused to the acute myelogenous leukemia 1 (AML1) transcription factor in t(8;21) AML, interacts via its zinc finger region with a conserved domain of the corepressors N-CoR and SMRT and recruits HDAC in vivo. The fusion protein AML1-ETO retains the ability of ETO to form stable complexes with N-CoR/SMRT and HDAC. Deletion of the ETO C terminus abolishes CoR binding and HDAC recruitment and severely impairs the ability of AML1-ETO to inhibit differentiation of hematopoietic precursors. These data indicate that formation of a stable complex with CoR-HDAC is crucial to the activation of the leukemogenic potential of AML1 by ETO and suggest that aberrant recruitment of corepressor complexes is a general mechanism of leukemogenesis.

Oligomerization of ETO is obligatory for corepressor interaction.

Nearly 40% of cases of acute myelogenous leukemia (AML) of the M2 subtype are due to a chromosomal translocation that combines a sequence-specific DNA binding protein, AML1, with a potent transcriptional repressor, ETO. ETO interacts with nuclear receptor corepressors SMRT and N-CoR, which recruit histone deacetylase to the AML1-ETO oncoprotein. SMRT-N-CoR interaction requires each of two zinc fingers contained in C-terminal Nervy homology region 4 (NHR4) of ETO. However, here we show that polypeptides containing NHR4 are insufficient for interaction with SMRT. NHR2 is also required for SMRT interaction and repression by ETO, as well as for inhibition of hematopoietic differentiation by AML1-ETO. NHR2 mediates oligomerization of ETO as well as AML1-ETO. Fusion of NHR4 polypeptide to a heterologous dimerization domain allows strong interaction with SMRT in vitro. These data support a model in which NHR2 and NHR4 have complementary functions in repression by ETO. NHR2 functions as an oligomerization domain bringing together NHR4 polypeptides that together form the surface required for high-affinity interaction with corepressors. As nuclear receptors also interact with corepressors as dimers, oligomerization may be a common mechanism regulating corepressor interactions.

Histone deacetylase-targeted treatment restores retinoic acid signaling and differentiation in acute myeloid leukemia.

Histone deacetylase (HDAC)-dependent transcriptional repression of the retinoic acid (RA)-signaling pathway underlies the differentiation block of acute promyelocytic leukemia. RA treatment relieves transcriptional repression and triggers differentiation of acute promyelocytic leukemia blasts, leading to disease remission. We report that transcriptional repression of RA signaling is a common mechanism in acute myeloid leukemias (AMLs). HDAC inhibitors restored RA-dependent transcriptional activation and triggered terminal differentiation of primary blasts from 23 AML patients. Accordingly, we show that AML1/ETO, the commonest AML-associated fusion protein, is an HDAC-dependent repressor of RA signaling. These findings relate alteration of the RA pathway to myeloid leukemogenesis and underscore the potential of transcriptional/differentiation therapy in AML.

Formation of PML/RAR alpha high molecular weight nuclear complexes through the PML coiled-coil region is essential for the PML/RAR alpha-mediated retinoic acid response.

Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukaemia (APL) patients by triggering terminal differentiation of neoplastic cells. RA-sensitivity in APL is mediated by its oncogenic protein, which results from the recombination of the PML and the RA receptor alpha (RAR alpha) genes (PML/RAR alpha fusion protein). Ectopic expression of PML/RAR alpha into haemopoietic cell lines results in increased response to RA-induced differentiation. By structure-function analysis of PML/RAR alpha-mediated RA-differentiation, we demonstrated that fusion of PML and RAR alpha sequences and integrity of the PML dimerization domain and of the RAR alpha DNA binding region are required for the effect of PML/RAR alpha on RA-differentiation. Indeed, direct fusion of the PML dimerization domain to the N- or C-terminal extremities of RAR alpha retained full biological activity. All the biologically active PML/RAR alpha mutants formed high molecular weight complexes in vivo. Functional analysis of mutations within the PML dimerization domain revealed that the capacity to form PML/RAR alpha homodimers, but not PML/RAR alpha-PML heterodimers, correlated with the RA-response. These results suggest that targeting of RAR alpha sequences by the PML dimerization domain and formation of nuclear PML/RAR alpha homodimeric complexes are crucial for the ability of PML/RAR alpha to mediate RA-response.

Common themes in the pathogenesis of acute myeloid leukemia.

The pathogenesis of acute myeloid leukemia is associated with the appearance of oncogenic fusion proteins generated as a consequence of specific chromosome translocations. Of the two components of each fusion protein, one is generally a transcription factor, whereas the other partner is more variable in function, but often involved in the control of cell survival and apoptosis. As a consequence, AML-associated fusion proteins function as aberrant transcriptional regulators that interfere with the process of myeloid differentiation, determine a stage-specific arrest of maturation and enhance cell survival in a cell-type specific manner. The abnormal regulation of transcriptional networks occurs through common mechanisms that include recruitment of aberrant co-repressor complexes, alterations in chromatin remodeling, and disruption of specific subnuclear compartments. The identification and analysis of common and specific target genes regulated by AML fusion proteins will be of fundamental importance for the full understanding of acute myeloid leukemogenesis and for the implementation of disease-specific drug design.

Terminal megakaryocytic differentiation of TF-1 cells is induced by phorbol esters and thrombopoietin and is blocked by expression of PML/RARalpha fusion protein.

We have analyzed the differentiation program of growth factor-dependent TF-1 erythroleukemia cells as well as clones with inducible expression of the APL-specific PML/RARalpha protein. We have shown that TF-1 cells may be induced to megakaryocytic differentiation by phorbol ester (phorbol dibutyrate, PDB) addition, particularly when combined with thrombopoietin (Tpo). RT-PCR studies showed that Tpo induces Tpo receptor (TpoR or c-mpl), whose expression was further potentiated by PDB addition. When the cells are induced with both PDB and Tpo erythropoietin receptor (EpoR) expression was inhibited. In the absence of Zn2+-induced PML/RARalpha expression, PDB and Tpo induced megakaryocytic differentiation of TF-1 MTPR clones as observed in 'wild-type' TF-1 cells. Conversely, when PML/RARalpha expression was induced by Zn2+, PDB and Tpo treatment of these clones caused only a reduced level of megakaryocytic differentiation. These observations indicate that: (1) TF-1 cells as well as other erythroleukemic cells, possess the capacity to differentiate to megakaryocytic cells when grown in the presence of protein kinase (PKC) activators and more efficiently when combined with Tpo; (2) the PML/RARalpha gene has a wide capacity to interfere with the program of hematopoietic differentiation, including megakaryocytic differentiation. Finally, we also observed that PML/RARalpha expression in TF-1 cells induces an up-modulation of interleukin-3 receptor, c-kit and c-mpl, a phenomenon which may offer these cells a growth advantage.

PINK1 protects against cell death induced by mitochondrial depolarization, by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage.

Mutations in the PINK1 gene are a frequent cause of autosomal recessive Parkinson's disease (PD). PINK1 encodes a mitochondrial kinase with neuroprotective activity, implicated in maintaining mitochondrial homeostasis and function. In concurrence with Parkin, PINK1 regulates mitochondrial trafficking and degradation of damaged mitochondria through mitophagy. Moreover, PINK1 can activate autophagy by interacting with the pro-autophagic protein Beclin-1. Here, we report that, upon mitochondrial depolarization, PINK1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to Beclin-1. PINK1-Bcl-xL interaction does not interfere either with Beclin-1 release from Bcl-xL or the mitophagy pathway; rather it protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL. Our data provide a functional link between PINK1, Bcl-xL and apoptosis, suggesting a novel mechanism through which PINK1 regulates cell survival. This pathway could be relevant for the pathogenesis of PD as well as other diseases including cancer.

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy.

Mutations in the PINK1 gene cause autosomal recessive Parkinson's disease. The PINK1 gene encodes a protein kinase that is mitochondrially cleaved to generate two mature isoforms. In addition to its protective role against mitochondrial dysfunction and apoptosis, PINK1 is also known to regulate mitochondrial dynamics acting upstream of the PD-related protein Parkin. Recent data showed that mitochondrial Parkin promotes the autophagic degradation of dysfunctional mitochondria, and that stable PINK1 silencing may have an indirect role in mitophagy activation. Here we report a new interaction between PINK1 and Beclin1, a key pro-autophagic protein already implicated in the pathogenesis of Alzheimer's and Huntington's diseases. Both PINK1 N- and C-terminal are required for the interaction, suggesting that full-length PINK1, and not its cleaved isoforms, interacts with Beclin1. We also demonstrate that PINK1 significantly enhances basal and starvation-induced autophagy, which is reduced by knocking down Beclin1 expression or by inhibiting the Beclin1 partner Vps34. A mutant, PINK1(W437X), interaction of which with Beclin1 is largely impaired, lacks the ability to enhance autophagy, whereas this is not observed for PINK1(G309D), a mutant with defective kinase activity but unaltered ability to bind Beclin1. These findings identify a new function of PINK1 and further strengthen the link between autophagy and proteins implicated in the neurodegenerative process.

Constitutive degradation of PML/RARalpha through the proteasome pathway mediates retinoic acid resistance.

PML/RARalpha is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RARalpha, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RARalpha protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RARalpha is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RARalpha protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RARalpha, but not RARalpha, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RARalpha protein may lead to RA resistance and that PML/RARalpha expression is crucial to convey RA sensitivity to APL cells.

PML/RAR alpha fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype.

The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RAR alpha fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RAR alpha mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RAR alpha alters the early hematopoietic program through N-CoR-dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells. (Blood. 2000;96:1531-1537)

Effects on differentiation by the promyelocytic leukemia PML/RARalpha protein depend on the fusion of the PML protein dimerization and RARalpha DNA binding domains.

The block of terminal differentiation is a prominent feature of acute promyelocytic leukemia (APL) and its release by retinoic acid correlates with disease remission. Expression of the APL-specific PML/RARalpha fusion protein in hematopoietic precursor cell lines blocks terminal differentiation, suggesting that PML/ RARalpha may have the same activity in APL blasts. We expressed different PML/RARalpha mutants in U937 and TF-1 cells and demonstrated that the integrity of the PML protein dimerization and RARalpha DNA binding domains is crucial for the differentiation block induced by PML/RARalpha, and that these domains exert their functions only within the context of the fusion protein. Analysis of the in vivo dimerization and cell localization properties of the PML/RARalpha mutants revealed that PML/RARalpha--PML and PML/RARalpha--RXR heterodimers are not necessary for PML/RARalpha activity on differentiation. We propose that a crucial mechanism underlying PML/RARalpha oncogenic activity is the deregulation of a transcription factor, RARalpha, through its fusion with the dimerization interface of another nuclear protein, PML.

Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARalpha fusion protein.

All-trans-retinoic acid (RA) treatment induces morphological remission in acute promyelocytic leukemia (APL) patients carrying the t(15;17) and expressing the PML/RARalpha product by inducing terminal differentiation of the leukemic clone. RA treatment induces downregulation of PML/RARalpha and reorganization of the PML-nuclear bodies. These events have been proposed to be essential for the induction of APL cell differentiation by RA. Here, we show that in the APL-derived NB4 cell line as well as in myeloid precursor U937 cells expressing the PML/RARalpha (U937/PR9) and in blasts from APL patients, the PML/RARalpha fusion protein is cleaved by a caspase 3-like activity induced by RA treatment. In fact, a caspase 3-like activity is detectable in PML/RARalpha expressing cells after RA treatment, and selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RARalpha deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the alpha-helix region of the PML component of the fusion protein. The extent of PML/RARalpha cleavage directly correlates with the ability of RA to restore the normal PML nuclear bodies (NBs) pattern. However, RA-induced differentiation is not prevented by the persistence of the fusion product and occurs in the absence of normally structured PML NBs. These results indicate that PML/RARalpha is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PML/RARalpha.

Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia.

The transforming proteins of acute promyelocytic leukaemias (APL) are fusions of the promyelocytic leukaemia (PML) and the promyelocytic leukaemia zinc-finger (PLZF) proteins with retinoic acid receptor-alpha (RARalpha). These proteins retain the RARalpha DNA- and retinoic acid (RA)-binding domains, and their ability to block haematopoietic differentiation depends on the RARalpha DNA-binding domain. Thus RA-target genes are downstream effectors. However, treatment with RA induces differentiation of leukaemic blast cells and disease remission in PML-RARalpha APLs, whereas PLZF-RARa APLs are resistant to RA. Transcriptional regulation by RARs involves modifications of chromatin by histone deacetylases, which are recruited to RA-target genes by nuclear co-repressors. Here we show that both PML-RARalpha and PLZF-RARalpha fusion proteins recruit the nuclear co-repressor (N-CoR)-histone deacetylase complex through the RARalpha CoR box. PLZF-RARalpha contains a second, RA-resistant binding site in the PLZF amino-terminal region. High doses of RA release histone deacetylase activity from PML-RARalpha, but not from PLZF-RARalpha. Mutation of the N-CoR binding site abolishes the ability of PML-RARalpha to block differentiation, whereas inhibition of histone deacetylase activity switches the transcriptional and biological effects of PLZF-RARalpha from being an inhibitor to an activator of the RA signalling pathway. Therefore, recruitment of histone deacetylase is crucial to the transforming potential of APL fusion proteins, and the different effects of RA on the stability of the PML-RARalpha and PLZF-RARalpha co-repressor complexes determines the differential response of APLs to RA.

Oligomerization of RAR and AML1 transcription factors as a novel mechanism of oncogenic activation.

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.