Coenzyme A precursors flow from mother to zygote and from microbiome to host.

Coenzyme A (CoA) is essential for metabolism and protein acetylation. Current knowledge holds that each cell obtains CoA exclusively through biosynthesis via the canonical five-step pathway, starting with pantothenate uptake. However, recent studies have suggested the presence of additional CoA-generating mechanisms, indicating a more complex system for CoA homeostasis. Here, we uncovered pathways for CoA generation through inter-organismal flows of CoA precursors. Using traceable compounds and fruit flies with a genetic block in CoA biosynthesis, we demonstrate that progeny survive embryonal and early larval development by obtaining CoA precursors from maternal sources. Later in life, the microbiome can provide the essential CoA building blocks to the host, enabling continuation of normal development. A flow of stable, long-lasting CoA precursors between living organisms is revealed. This indicates the presence of complex strategies to maintain CoA homeostasis.

Vps13 is required for timely removal of nurse cell corpses.

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionarily conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. It is expressed in late nurse cells, and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells.

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle.

Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis.IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.

Early detection and evolution of preleukemic clones in therapy-related myeloid neoplasms following autologous SCT.

Therapy-related myeloid neoplasms (tMNs) are severe adverse events that can occur after treatment with autologous hematopoietic stem cell transplantation (ASCT). This study aimed to investigate the development of tMNs following ASCT at the molecular level by whole-exome sequencing (WES) and targeted deep sequencing (TDS) in sequential (pre-) tMN samples. WES identified a significantly higher number of mutations in tMNs as compared with de novo myelodysplastic syndrome (MDS) (median 27 vs 12 mutations; P = .001). The mutations found in tMNs did not carry a clear aging-signature, unlike the mutations found in de novo MDS, indicating a different mutational mechanism. In some patients, tMN mutations were identified in both myeloid and T cells, suggesting that tMNs may originate from early hematopoietic stem cells (HSCs). However, the mutational spectra of tMNs and the preceding malignancies did not overlap, excluding common ancestry for these malignancies. By use of TDS, tMN mutations were identified at low variant allele frequencies (VAFs) in transplant material in 70% of the patients with tMNs. Reconstruction of clonal patterns based on VAFs revealed that premalignant clones can be present more than 7 years preceding a tMN diagnosis, a finding that was confirmed by immunohistochemistry on bone marrow biopsies. Our results indicate that tMN development after ASCT originates in HSCs bearing (pre-)tMN mutations that are present years before disease onset and that post-ASCT treatment can influence the selection of these clones. Early detection of premalignant clones and monitoring of their evolutionary trajectory may help to predict the development of tMNs and guide early intervention in the future.

The TAK1-NF-κB axis as therapeutic target for AML.

Development and maintenance of leukemia can be partially attributed to alterations in (anti)-apoptotic gene expression. Genome-wide transcriptome analyses revealed that 89 apoptosis-associated genes were differentially expressed between patient acute myeloid leukemia (AML) CD34(+) cells and normal bone marrow (NBM) CD34(+) cells. Among these, transforming growth factor-ß activated kinase 1 (TAK1) was strongly upregulated in AML CD34(+) cells. Genetic downmodulation or pharmacologic inhibition of TAK1 activity strongly impaired primary AML cell survival and cobblestone formation in stromal cocultures. TAK1 inhibition was mainly due to blockade of the nuclear factor κB (NF-κB) pathway, as TAK1 inhibition resulted in reduced levels of P-IκBα and p65 activity. Overexpression of a constitutive active variant of NF-κB partially rescued TAK1-depleted cells from apoptosis. Importantly, NBM CD34(+) cells were less sensitive to TAK1 inhibition compared with AML CD34(+) cells. Knockdown of TAK1 also severely impaired leukemia development in vivo and prolonged overall survival in a humanized xenograft mouse model. In conclusion, our results indicate that TAK1 is frequently overexpressed in AML CD34(+) cells, and that TAK1 inhibition efficiently targets leukemic stem/progenitor cells in an NF-κB-dependent manner.

Impact of genetic variation in the SMIM1 gene on Vel expression levels.

BACKGROUND: Serologic determination of the Vel- phenotype is challenging due to variable Vel expression levels. In this study we investigated the genetic basis for weak Vel expression levels and developed a high-throughput genotyping assay to detect Vel- donors. STUDY DESIGN AND METHODS: In 548 random Caucasian and 107 Vel+(w) donors genetic variation in the SMIM1 gene was studied and correlated to Vel expression levels. A total of 3366 Caucasian, 621 black, and 333 Chinese donors were screened with a high-throughput genotyping assay targeting the SMIM1*64_80del allele. RESULTS: The Vel+(w) phenotype is in most cases caused by the presence of one SMIM1 allele carrying the major allele of the rs1175550 SNP in combination with a SMIM1*64_80del allele or in few cases caused by the presence of the SMIM1*152T>A or SMIM1*152T>G allele. In approximately 6% of Vel+(w) donors genetic factors in SMIM1 could not explain the weak expression. We excluded the possibility that lack of expression of another blood group system was correlated with weak Vel expression levels. Furthermore, using a high-throughput Vel genotyping assay we detected two Caucasian Vel- donors. CONCLUSION: Weak Vel expression levels are caused by multiple genetic factors in SMIM1 and probably also by other genetic or environmental factors. Due to the variation in Vel expression levels, serologic determination of the Vel- phenotype is difficult and a genotyping assay targeting the c.64_80del deletion in SMIM1 should be used to screen donors for the Vel- phenotype.

Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome.

A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.

Cells are constantly perceiving and responding to changes in their surroundings, and challenging conditions such as extreme heat or toxic chemicals can put cells under stress. When this happens, protein production can be affected. Proteins are long chains of chemical building blocks called amino acids, and they can only perform their roles if they fold into the right shape. Some proteins fold easily and remain folded, but others can be unstable and often become misfolded. Unfolded proteins can become a problem because they stick to each other, forming large clumps called aggregates that can interfere with the normal activity of cells, causing damage. The causes of stress that have a direct effect on protein folding are called proteotoxic stresses, and include, for example, high temperatures, which make proteins more flexible and unstable, increasing their chances of becoming unfolded. To prevent proteins becoming misfolded, cells can make 'protein chaperones', a type of proteins that help other proteins fold correctly and stay folded. The production of protein chaperones often increases in response to proteotoxic stress. However, there are other types of stress too, such as genotoxic stress, which damages DNA. It is unclear what effect genotoxic stress has on protein folding. Huiting et al. studied protein folding during genotoxic stress in human cells grown in the lab. Stress was induced by either blocking the proteins that repair DNA or by 'trapping' the proteins that release DNA tension, both of which result in DNA damage. The analysis showed that, similar to the effects of proteotoxic stress, genotoxic stress increased the number of proteins that aggregate, although certain proteins formed aggregates even without stress, particularly if they were common and relatively unstable proteins. Huiting et al.'s results suggest that aggregation increases in cells under genotoxic stress because the cells fail to produce enough chaperones to effectively fold all the proteins that need it. Indeed, Huiting et al. showed that aggregates contain many proteins that rely on chaperones, and that increasing the number of chaperones in stressed cells reduced protein aggregation. This work shows that genotoxic stress can affect protein folding by limiting the availability of chaperones, which increases protein aggregation. Remarkably, there is a substantial overlap between proteins that aggregate in diseases that affect the brain ­ such as Alzheimer's disease ­ and proteins that aggregate after genotoxic stress. Therefore, further research could focus on determining whether genotoxic stress is involved in the progression of these neurological diseases.

Coenzyme A levels influence protein acetylation, CoAlation and 4'-phosphopantetheinylation: Expanding the impact of a metabolic nexus molecule.

Coenzyme A (CoA) is a key molecule in cellular metabolism including the tricarboxylic acid cycle, fatty acid synthesis, amino acid synthesis and lipid metabolism. Moreover, CoA is required for biological processes like protein post-translational modifications (PTMs) including acylation. CoA levels affect the amount of histone acetylation and thereby modulate gene expression. A direct influence of CoA levels on other PTMs, like CoAlation and 4'-phosphopantetheinylation has been relatively less addressed and will be discussed here. Increased CoA levels are associated with increased CoAlation, whereas decreased 4'-phosphopantetheinylation is observed under circumstances of decreased CoA levels. We discuss how these two PTMs can positively or negatively influence target proteins depending on CoA levels. This review highlights the impact of CoA levels on post-translational modifications, their counteractive interplay and the far-reaching consequences thereof.

The Hippo signaling pathway effector YAP promotes salivary gland regeneration after injury.

Salivary glands are damaged by radiotherapy for head and neck cancers, which often culminates in radiation-induced hyposalivation and xerostomia that may be permanent. Here, we identified a central role for YAP in the regenerative response of the salivary gland. Activation of the Hippo signaling pathway inhibits the phosphorylation of YAP, leading to its nuclear translocation and transcriptional activity. Using mice with salivary gland injury induced by surgical ligation and salivary gland­derived organoids, we found that YAP nuclear localization in the salivary gland epithelium changed dynamically between homeostasis and regeneration. Whereas local injury had no effect on nuclear YAP localization in saliva-producing acinar cells, it triggered nuclear accumulation of YAP in saliva-transporting ductal cells. Injury also stimulated the proliferation of ductal cells, which were mainly quiescent under homeostatic conditions and in nonregenerating areas distal to the injury site, thus enabling salivary gland regeneration. Overexpressing YAP or driving YAP nuclear translocation by inhibiting upstream Hippo pathway kinases increased the capacity of mouse and human salivary gland cells, including human cells that had been irradiated, to form lobed organoids in vitro. Our results identify a YAP-driven regeneration program in salivary gland ductal cells that could be used to promote salivary gland regeneration after irradiation-induced damage.

Ex vivo assays to study self-renewal and long-term expansion of genetically modified primary human acute myeloid leukemia stem cells.

With the emergence of the concept of the leukemia stem cell, assays to study them remain pivotal in understanding (leukemic) stem cell biology. Although the in vivo NOD-SCID xenotransplantation model is still the favored model of choice in most cases, this system has some limitations as well, such as its cost-effectiveness, duration, and the lack of engraftability of cells from subsets of acute myeloid leukemia (AML) patients. Here, we have described an ex vivo bone marrow stromal coculture system in which CD34(+) cells, but not CD34(-) cells, from the bone marrow or peripheral blood of AML patients can give rise to long-term cultures (LTC) that can be maintained for over 20 weeks. Long-term expansion is associated with the formation of leukemic cobblestone area (L-CA) formation underneath the stroma. Self-renewal within these L-CAs can be determined by sequential passaging of these L-CAs onto new MS5 stromal layers, which results in the generation of second, third, and fourth L-CAs that are able to sustain long-term expansion and generate high numbers of immature undifferentiated suspension cells. Furthermore, we have optimized lentiviral transduction procedures in order to stably express genes of interest or stably downmodulate genes using RNAi in AML CD34(+) cells, and this method has also been described here. Together, these tools should allow a further molecular elucidation of derailed signal transduction in AML stem cells.

Mucin1 expression is enriched in the human stem cell fraction of cord blood and is upregulated in majority of the AML cases.

OBJECTIVE: Mucin1 is a membrane glycoprotein that is overexpressed in a variety of human cancers. Here, we analyzed the role of Mucin1 in human hematopoietic stem/progenitor cells as well as in acute myeloid leukemia (AML) cells. MATERIALS AND METHODS: Mucin1 expression was determined within the normal stem cell and progenitor compartment, as well as in the AML CD34+ and CD34- subfractions of patient samples. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays in limiting dilution and progenitor frequencies in colony-forming cell (CFC) assays in methylcellulose, and consequences of elevated Mucin1 expression were studied using retroviral overexpression systems in cord blood (CB) CD34+ cells. RESULTS: Ten percent of CB and 5% of peripheral blood CD34+ cells expressed Mucin1. Retroviral overexpression of Mucin1 in CB CD34+ cells resulted in elevated stem cell and progenitor frequencies as determined in LTC-IC and CFC assays without affecting differentiation, which coincided with increased proliferation. Overexpression of intercellular adhesion molecule-1, a ligand for Mucin1, in MS5 stromal cells further increased LTC-IC frequencies. Mucin1 overexpression was associated with increased nuclear factor-kappaB p50 nuclear translocation, suggesting that Mucin1-induced phenotypes involve increased cell survival mechanisms. Finally, we observed increased Mucin1 expression in 70% of the AML cases (n=24), suggesting that elevated Mucin1 levels might be involved in regulating the proliferative potential of the immature leukemic compartment as well. CONCLUSIONS: Our data indicate that hematopoietic stem cells as well as CD34+ AML subfractions are enriched for Mucin1 expression, and that overexpression of Mucin1 in CB cells is sufficient to increase both progenitor and LTC-IC frequencies.

Differential redox-regulation and mitochondrial dynamics in normal and leukemic hematopoietic stem cells: A potential window for leukemia therapy.

The prognosis for many patients with acute myeloid leukemia (AML) is poor, mainly due to disease relapse driven by leukemia stem cells (LSCs). Recent studies have highlighted the unique metabolic properties of LSCs, which might represent opportunities for LSC-selective targeting. LSCs characteristically have low levels of reactive oxygen species (ROS), which apparently result from a combination of low mitochondrial activity and high activity of ROS-removing pathways such as autophagy. Due to this low activity, LSCs are highly dependent on mitochondrial regulatory mechanisms. These include the anti-apoptotic protein BCL-2, which also has crucial roles in regulating the mitochondrial membrane potential, and proteins involved in mitophagy. Here we review the different pathways that impact mitochondrial activity and redox-regulation, and highlight their relevance for the functionality of both HSCs and LSCs. Additionally, novel AML therapy strategies that are based on interference with those pathways, including the promising BCL-2 inhibitor Venetoclax, are summarized.

Transcriptional regulators CITED2 and PU.1 cooperate in maintaining hematopoietic stem cells.

Reduced expression of the transcription factor PU.1 is frequently associated with development of acute myeloid leukemia (AML), whereas elevated levels of CITED2 (CBP/p300-interacting-transactivator-with-an-ED-rich-tail 2) enhance maintenance of both normal and leukemic hematopoietic stem and progenitor cells (HSPCs). Recent findings indicate that PU.1 and CITED2 act in the same gene regulatory network. We therefore examined a potential synergistic effect of simultaneous PU.1 downregulation and CITED2 upregulation on stem cell biology and AML pathogenesis. We found that simultaneous PU.1/CITED2 deregulation in human CD34+ cord blood (CB) cells, as well as CITED2 upregulation in preleukemic murine PU.1-knockdown (PU.1KD/KD) bone marrow cells, significantly increased the maintenance of HSPCs compared with the respective deregulation of either factor alone. Increased replating capacity of PU.1KD/KD/CITED2 cells in in vitro assays eventually resulted in outgrowth of transformed cells, while upregulation of CITED2 in PU.1KD/KD cells enhanced their engraftment in in vivo transplantation studies without affecting leukemic transformation. Transcriptional analysis of CD34+ CB cells with combined PU.1/CITED2 alterations revealed a set of differentially expressed genes that highly correlated with gene signatures found in various AML subtypes. These findings illustrate that combined PU.1/CITED2 deregulation induces a transcriptional program that promotes HSPC maintenance, which might be a prerequisite for malignant transformation.

CoA-dependent activation of mitochondrial acyl carrier protein links four neurodegenerative diseases.

PKAN, CoPAN, MePAN, and PDH-E2 deficiency share key phenotypic features but harbor defects in distinct metabolic processes. Selective damage to the globus pallidus occurs in these genetic neurodegenerative diseases, which arise from defects in CoA biosynthesis (PKAN, CoPAN), protein lipoylation (MePAN), and pyruvate dehydrogenase activity (PDH-E2 deficiency). Overlap of their clinical features suggests a common molecular etiology, the identification of which is required to understand their pathophysiology and design treatment strategies. We provide evidence that CoA-dependent activation of mitochondrial acyl carrier protein (mtACP) is a possible process linking these diseases through its effect on PDH activity. CoA is the source for the 4'-phosphopantetheine moiety required for the posttranslational 4'-phosphopantetheinylation needed to activate specific proteins. We show that impaired CoA homeostasis leads to decreased 4'-phosphopantetheinylation of mtACP. This results in a decrease of the active form of mtACP, and in turn a decrease in lipoylation with reduced activity of lipoylated proteins, including PDH. Defects in the steps of a linked CoA-mtACP-PDH pathway cause similar phenotypic abnormalities. By chemically and genetically re-activating PDH, these phenotypes can be rescued, suggesting possible treatment strategies for these diseases.

CBX7 Induces Self-Renewal of Human Normal and Malignant Hematopoietic Stem and Progenitor Cells by Canonical and Non-canonical Interactions.

In this study, we demonstrate that, among all five CBX Polycomb proteins, only CBX7 possesses the ability to control self-renewal of human hematopoietic stem and progenitor cells (HSPCs). Xenotransplantation of CBX7-overexpressing HSPCs resulted in increased multi-lineage long-term engraftment and myelopoiesis. Gene expression and chromatin analyses revealed perturbations in genes involved in differentiation, DNA and chromatin maintenance, and cell cycle control. CBX7 is upregulated in acute myeloid leukemia (AML), and its genetic or pharmacological repression in AML cells inhibited proliferation and induced differentiation. Mass spectrometry analysis revealed several non-histone protein interactions between CBX7 and the H3K9 methyltransferases SETDB1, EHMT1, and EHMT2. These CBX7-binding proteins possess a trimethylated lysine peptide motif highly similar to the canonical CBX7 target H3K27me3. Depletion of SETDB1 in AML cells phenocopied repression of CBX7. We identify CBX7 as an important regulator of self-renewal and uncover non-canonical crosstalk between distinct pathways, revealing therapeutic opportunities for leukemia.

Ring sideroblasts in AML are associated with adverse risk characteristics and have a distinct gene expression pattern.

Ring sideroblasts (RS) emerge as result of aberrant erythroid differentiation leading to excessive mitochondrial iron accumulation, a characteristic feature for myelodysplastic syndromes (MDS) with mutations in the spliceosome gene SF3B1. However, RS can also be observed in patients diagnosed with acute myeloid leukemia (AML). The objective of this study was to characterize RS in patients with AML. Clinically, RS-AML is enriched for ELN adverse risk (55%). In line with this finding, 35% of all cases had complex cytogenetic aberrancies, and TP53 was most recurrently mutated in this cohort (37%), followed by DNMT3A (26%), RUNX1 (25%), TET2 (20%), and ASXL1 (19%). In contrast to RS-MDS, the incidence of SF3B1 mutations was low (8%). Whole-exome sequencing and SNP array analysis on a subset of patients did not uncover a single genetic defect underlying the RS phenotype. Shared genetic defects between erythroblasts and total mononuclear cell fraction indicate common ancestry for the erythroid lineage and the myeloid blast cells in patients with RS-AML. RNA sequencing analysis on CD34+ AML cells revealed differential gene expression between RS-AML and non RS-AML cases, including genes involved in megakaryocyte and erythroid differentiation. Furthermore, several heme metabolism-related genes were found to be upregulated in RS- CD34+ AML cells, as was observed in SF3B1mut MDS. These results demonstrate that although the genetic background of RS-AML differs from that of RS-MDS, they have certain downstream effector pathways in common.

Establishing long-term cultures with self-renewing acute myeloid leukemia stem/progenitor cells.

OBJECTIVE: With the emergence of the concept of the leukemia stem cell, assays to study them remain pivotal in understanding (leukemic) stem cell biology. METHODS: We have cultured acute myeloid leukemia CD34(+) cells on bone marrow stroma. Long-term expansion was monitored and self-renewal was addressed by replating of Leukemic-cobblestone area-forming cells (L-CAs). Also, lentiviral vectors were generated that could target L-CAs. RESULTS: A strong expansion was observed in about 75% of the acute myeloid leukemia cases (n = 30) and long-term cultures could be maintained for up to 24 weeks on MS5 bone marrow stromal cells. Cells that were able to initiate leukemic cobblestone areas resided in the CD34(+) population and were absent from the CD34(-) population. Self-renewal within these L-CAs was determined by sequential passaging of these L-CAs onto new MS5 stromal layers, which resulted in the generation of second, third, and fourth L-CAs, which were able to sustain long-term expansion and generated high numbers of immature undifferentiated suspension cells. CD34(+) cells that were able to initiate long-term cultures all coexpressed MEIS1 and HOXA9, and expressed elevated BMI1 levels. CONCLUSION: We present a novel long-term leukemic stem/progenitor assay in which new drugs can be tested and in which genes can be overexpressed or downmodulated using a lentiviral approach in order to obtain more insight into the process of leukemic transformation and self-renewal.

CITED2 affects leukemic cell survival by interfering with p53 activation.

CITED2 (CBP/p300-interacting-transactivator-with-an-ED-rich-tail 2) is a regulator of the acetyltransferase CBP/p300 and elevated CITED2 levels are shown in a number of acute myeloid leukemia (AML). To study the in vivo role of CITED2 in AML maintenance, AML cells were transduced with a lentiviral construct for RNAi-mediated knockdown of CITED2. Mice transplanted with CITED2-knockdown AML cells (n=4) had a significantly longer survival compared to mice transplanted with control AML cells (P<0.02). In vitro, the reduction of CITED2 resulted in increased p53-mediated apoptosis and CDKN1A expression, whereas BCL2 levels were reduced. The activation of p53 upon CITED2 knockdown is not a direct consequence of increased CBP/p300-activity towards p53, since no increased formation of CBP/p300/p53 complexes was demonstrated and inhibition of CBP/p300-activity could not rescue the phenotype of CITED2-deficient cells. Instead, loss of CITED2 had an inhibitory effect on the AKT-signaling pathway, which was indicated by decreased levels of phosphorylated AKT and altered expression of the AKT-pathway regulators PHLDA3 and SOX4. Notably, simultaneous upregulation of BCL2 or downregulation of the p53-target gene PHLDA3 rescued the apoptotic phenotype in CITED2-knockdown cells. Furthermore, knockdown of CITED2 led to a decreased interaction of p53 with its inhibitor MDM2, which results in increased amounts of total p53 protein. In summary, our data indicate that CITED2 functions in pathways regulating p53 activity and therefore represents an interesting target for AML therapy, since de novo AML cases are characterized by an inactivation of the p53 pathway or deregulation of apoptosis-related genes.

Constitutive activation of NF-kappa B is not sufficient to disturb normal steady-state hematopoiesis.

Since nuclear factor-kappaB (NF-kappaB) is frequently activated in acute myeloid leukemia, we questioned whether active NF-kappaB can affect the cellular properties of cord blood CD34+ cells. The results demonstrated that NF-kappaB activation did not influence growth or differentiation properties of these cells. Furthermore, NF-kappaB activation was not sufficient to induce changes in stem- and progenitor cell numbers.

Oncogenic Ras blocks transforming growth factor-beta-induced cell-cycle arrest by degradation of p27 through a MEK/Erk/SKP2-dependent pathway.

OBJECTIVE: To examine whether oncogenic Ras affects transforming growth factor (TGF)-beta-mediated cell-cycle arrest in hematopoietic cells and the downstream signal transduction pathway involved in the interference with TGF-beta-induced cell-cycle arrest. MATERIALS AND METHODS: Two leukemic cell lines bearing N-Ras(L61) mutations; HL-60 and TF-1, and the M1 cell line with wt Ras were investigated for their response to TGF-beta. Signal transduction inhibitors, overexpression and RNA interference studies were performed to investigate the involvement of the various proteins. RESULTS: Although TGF-beta signal transduction was not affected, G0-G1 arrest was absent in HL-60 and TF-1 cells due to the absence of p27. Overexpression of p27 restored TGF-beta-induced cell-cycle arrest, as well as interfering in Ras-mediated signaling. The farnesyl transferase inhibitor L744832 and the MEK inhibitor U0126 both restored p27 levels and cell-cycle arrest in response to TGF-beta. The absence of p27 protein is due to elevated levels of the ubiquitin ligase SKP2, which complexes with and targets p27 for degradation. RNA interference for SKP2 and treatment of these cells with the proteasome inhibitor MG132 restored p27 levels, corresponding with decreasing SKP2 levels after interfering in N-Ras signal transduction. P27, phosphorylated at threonine 187, is nuclear localized in N-Ras-containing cells. Mutation of this residue to alanine rendered p27 insensitive to degradation. CONCLUSION: N-Ras(L61) transformed cells lack a G0-G1 arrest upon TGF-beta treatment due to absence of p27. p27 is degraded through a MapK-, and SKP2-dependent pathway. Overexpression of p27 results in restoration of cell-cycle arrest upon TGF-beta treatment.