Somatic chromosomal deletions in cancer are thought to indicate the location of tumour suppressor genes, by which a complete loss of gene function occurs through biallelic deletion, point mutation or ...epigenetic silencing, thus fulfilling Knudson's two-hit hypothesis. In many recurrent deletions, however, such biallelic inactivation has not been found. One prominent example is the 5q super(-) syndrome, a subtype of myelodysplastic syndrome characterized by a defect in erythroid differentiation. Here we describe an RNA-mediated interference (RNAi)-based approach to discovery of the 5q super(-) disease gene. We found that partial loss of function of the ribosomal subunit protein RPS14 phenocopies the disease in normal haematopoietic progenitor cells, and also that forced expression of RPS14 rescues the disease phenotype in patient-derived bone marrow cells. In addition, we identified a block in the processing of pre-ribosomal RNA in RPS14-deficient cells that is functionally equivalent to the defect in Diamond-Blackfan anaemia, linking the molecular pathophysiology of the 5q super(-) syndrome to a congenital syndrome causing bone marrow failure. These results indicate that the 5q super(-) syndrome is caused by a defect in ribosomal protein function and suggest that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent work (Ebert et al, Nature 2008, Jan 17:335-9) has shown that ribosomal protein S14 (RPS14) is underexpressed in myelodysplasia (MDS) with deletion of chromosome 5q and plays a role in its ...pathogenesis. The role of ribosomal proteins in other more common subtypes of MDS is unknown. We conducted a meta-analytical comparison of gene expression profiles of 60 cases of non 5q- MDS CD34+ cells with 52 normal CD34+ profiles. These datasets were obtained from seven independent studies from NCBI's GEO database. The data was integrated based on UniGene IDs and were quantile normalized to ensure cross-study comparability. (Based on our previous approach; Sohal et al PLOS One, 2008, Zhou et al, Blood, 2008). Using significance analysis of microarrays (SAM) and a false discovery rate (FDR) of just 0.04%, we found that ribosomal protein were the class of genes that were most significantly altered in MDS. We observed that RPL35a, RPS9, RPL10, RPL22, RPS14, RPS10, RPS15a, RPS24, RPL24, RPL36, RPL21, RPL23 were strikingly downregulated in non-5q- MDS CD34+ cells. To determine if these alterations were a result of changes in DNA copy numbers of these genes, we examined 20 MDS samples by high resolution array comparative genome hybridization (aCGH) performed on Nimblegen whole genome tiling arrays. aCGH at 6kb resolution revealed deletions in RPL14, RPL22, RPL36, RPS10, RPS5 and even RPS14 in distinct selected cases of non 5q- MDS. These small deletions, which were not identifiable by traditional karyotyping methods, may be putative mechanisms of ribosomal protein downregulation and ultimately, in MDS development and/or progression. Since MDS is also characterized by aberrant epigenetic silencing of genes, we next examined the methylation status of ribosomal gene promoters by high resolution global DNA methylation profiling by using the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)). This assay uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. While there were sporadic changes in some patients, we did not observe any consistently significant changes in methylation of these ribosomal gene promoters on comparison with normal anemic controls.
In summary, we show novel widespread alterations in ribosomal protein expression in MDS, at least some of which are associated with genomic deletions. These findings illustrate the applicability of meta-analytical genomic approaches in a heterogeneous disease such as MDS. Most importantly, our data points to the dramatic role of alteration in the protein translational machinery in pathogenesis of MDS.
We report here that haploinsufficiency of the ribosomal protein encoding RPS14 gene causes the characteristic hematologic phenotype that defines the 5q- syndrome. The 5q- syndrome, a subtype of ...myelodysplastic syndrome (MDS), was first described in 1974 as a distinct clinical entity associated with deletions of the long arm of chromosome 5. Since that time, the common deleted region has been narrowed in search of the causal gene within the region. However, no biallelic inactivation events have been identified for any of the 41 genes within the critical region, suggesting that a haploinsufficiency mechanism may explain the disease phenotype. We addressed this question through a functional genomic approach, utilizing systematic RNA interference (RNAi) to interrogate the function of each gene in the common deleted region. We introduced 3–5 unique, lentivirally expressed short hairpin RNAs (shRNAs) targeting each of the 41 genes in the region into normal CD34+ human bone marrow hematopoietic progenitor cells and assayed the effects of each shRNA on hematopoietic differentiation. We found that knock-down of a single gene, RPS14, recapitulated the phenotype of the 5q- syndrome: namely, a block in erythroid differentiation (leading to erythroid cell apoptosis) with relative preservation of megakaryocyte differentiation as measured by FACS analysis. Importantly, forced expression of an RPS14 cDNA in primary bone marrow cells from patients with the 5q- syndrome rescued the phenotype, yet had no effect on cells from MDS patients without 5q deletions. In addition, we found that RPS14 haploinsufficiency caused a block in the processing of pre-ribosomal RNA and in the formation of the 40S ribosomal subunit. This ribosomal processing defect is highly analogous to the functional defect seen in Diamond Blackfan Anemia (also characterized by an erythroid differentiation defect and predisposition to AML), thereby establishing an unexpected link between the molecular pathophysiology of acquired 5q- syndrome and congenital bone marrow failure syndromes. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function, highlighting the importance of translational control in hematologic malignancy. The results further suggest that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.
Vidaza and Thalidomide were administered to 29 patients with MDS or AML. Vidaza was given at a dose of 75mg/kg subq X 5 days q28 days and Thalidomide starting at 50mg/day and increasing to 100mg. ...Therapy was well tolerated. Median age was 70 years, and there were 16 males. Two patients had RA, 2 RARS, 9 RAEB, 4 CMMoL, 10 AML and 2 Unknowns. According to IPSS, 1 had low, 7 had Int-1, 5 had Int-2 and 4 had high risk disease, and 2 unclassified and 10 had AML. Eleven patients had normal, 14 abnormal and 4 unknown cytogenetics. Hematologic improvement (HI) was seen in 14 and stable disease in 6 while 5 had disease progression. cDNA from pre-therapy BM mononuclear cells of 28/29 patients was hybridized to Affymetrix HG-U133AA microarrays and data was analyzed using the GenePattern software package. Marker genes were selected using ClassNeighbors algorithm with the signal-to-noise metric. When comparing resistant disease (RD) patients with HI (6) and others (2), of the 40 top marker genes over-expressed in the RD group, 21 are directly associated with cellular proliferation and can be divided into 3 groups. Group I (cell cycle) consisted of 9 genes; BUB1B, CCNA2, TMPO, CDC2, CCNB1, UBE2C, TTK, CDC20 and MCM5. Group II (replication repair) had 5 genes; TOP2A, RRM2, RFC3, FEN1, RNAH2A. Group III (spindle/chromosome structure) had 7; KIF2C, NUSAP1, CENPF, MK167, SPAG5, CENPA and STK6. Over-expression of these genes would indicate rapid cellular proliferation which may be an underlying cause of drug resistance. These results are especially striking since the treatment regimen consisted of a combination of two drugs, Vidaza and thalidomide, which purportedly act through different mechanisms. We conclude that the RD patients share a molecular signature which is also consistent with a demonstrated mechanism of cell cycle resistance, and underscores the future clinical usefulness of this methodology.
The VHL tumor suppressor protein (pVHL) is part of an E3 ubiquitin ligase that targets HIF for destruction. pVHL-defective renal carcinoma cells exhibit increased NF-κB activity but the mechanism is ...unclear. NF-κB affects tumorigenesis and therapeutic resistance in some settings. We found that pVHL associates with the NF-κB agonist Card9 but does not target Card9 for destruction. Instead, pVHL serves as an adaptor that promotes the phosphorylation of the Card9 C-terminus by CK2. Elimination of these sites markedly enhanced Card9's ability to activate NF-κB in
VHL
+/+ cells and Card9 siRNA normalized NF-κB activity in
VHL
−/− cells and restored their sensitivity to cytokine-induced apoptosis. Furthermore, downregulation of Card9 in
VHL
−/− cancer cells reduced their tumorigenic potential. Therefore pVHL can serve as an adaptor for both an ubiquitin conjugating enzyme and for a kinase. The latter activity, which promotes Card9 phosphorylation, links pVHL to control of NF-κB activity and tumorigenesis.
Polycythemia vera, essential thrombocythemia and primary myelofibrosis are myeloproliferative neoplasms (MPN) characterized by multilineage clonal hematopoiesis
1
–
5
. Given that the identical ...somatic activating mutation in the
JAK2
tyrosine kinase gene (
JAK2
V617F
) is observed in most individuals with polycythemia vera, essential thrombocythemia and primary myelofibrosis
6
–
10
, there likely are additional genetic events that contribute to the pathogenesis of these phenotypically distinct disorders. Moreover, family members of individuals with MPN are at higher risk for the development of MPN, consistent with the existence of MPN predisposition loci
11
. We hypothesized that germline variation contributes to MPN predisposition and phenotypic pleiotropy. Genome-wide analysis identified an allele in the
JAK2
locus (rs10974944) that predisposes to the development of
JAK2
V617F
-positive MPN, as well as three previously unknown MPN modifier loci. We found that
JAK2
V617F
is preferentially acquired in cis with the predisposition allele. These data suggest that germline variation is an important contributor to MPN phenotype and predisposition.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Lineage specification is a critical issue in developmental and regenerative biology. We hypothesized that microRNAs (miRNAs) are important participants in those processes and used the poorly ...understood regulation of megakaryocyte-erythrocyte progenitors (MEPs) in hematopoiesis as a model system. We report here that miR-150 modulates lineage fate in MEPs. Using a novel methodology capable of profiling miRNA expression in small numbers of primary cells, we identify miR-150 as preferentially expressed in the megakaryocytic lineage. Through gain- and loss-of-function experiments, we demonstrate that miR-150 drives MEP differentiation toward megakaryocytes at the expense of erythroid cells in vitro and in vivo. Moreover, we identify the transcription factor MYB as a critical target of miR-150 in this regulation. These experiments show that miR-150 regulates MEP fate, and thus establish a role for miRNAs in lineage specification of mammalian multipotent cells.
Several lines of evidence indicate that the pharmacological activation of fetal hemoglobin is an effective therapy for sickle cell anemia and beta thalassemia, but novel treatments for these diseases ...are needed. We developed and validated a high throughput assay to detect differential regulation of the globin genes and utilized this assay in a small molecule screen to identify novel compounds that increase the relative expression of gamma globin. In our assay, transcripts for the alpha, beta, delta, epsilon, gamma, theta, and zeta globin genes are amplified by multiplexed ligation-mediated PCR. Labeled amplicons are captured on different fluorescent microspheres using molecular barcodes, and the relative abundance of labeled amplicons is detected by high speed flow cytometry. To recapitulate the activity of compounds in the bone marrow of patients as accurately as possible, the screen was performed using primary human erythroid progenitor cells cultured in vitro. The assay was adapted to 384-well format with robotic liquid handling. In validation studies, the assay detected the expected increases in globin gene expression during erythroid differentiation, increased gamma globin expression in umbilical cord blood progenitor cells, and increased gamma globin expression in cells treated with known inducers of fetal hemoglobin including hydroxyurea and sodium butyrate. We screened a library of 1040 known bioactive compounds, 75% of which are FDA approved drugs, and a library of 600 compounds produced by diversity oriented synthesis that have been shown to inhibit histone deacetylase (HDAC) activity. In the screen, we rediscovered previously identified globin gene regulators, further validating our globin assay. For example, corticosteroids, known activators of fetal hemoglobin, increased the relative expression of gamma globin. Thyroid hormone specifically increased expression of delta globin, consistent with clinical observations that hemoglobin A2 levels are increased in hyperthyroidism and decreased in hypothyroidism. We identified ten novel compounds from the diversity oriented synthesis library that powerfully induce expression of the gamma globin gene relative to beta globin. Moreover, HDAC inhibition reversed the ontogeny of globin gene expression, coordinately increasing expression of fetal and embryonic relative to the adult globin genes. Relative to beta globin gene expression, gamma and epsilon globin were induced while delta globin was unaffected by HDAC inhibitors; relative to alpha globin expression, zeta globin was increased and theta globin was unaffected. The identification of compounds that differentially regulate globin gene expression may provide lead compounds for the development of novel therapies for sickle cell disease and beta thalassemia and may help elucidate the molecular events underlying switching of the globin genes during normal development.
Activating mutations in the juxtamembrane domain or the activation loop of the receptor tyrosine kinase FLT3 occur in many cases of acute myeloid leukemia (AML), but it is not known whether genomic ...alterations outside these regions contribute to leukemogenesis. High-throughput DNA sequence analysis has provided insights into the mutational profiles of various cancers and represents a promising strategy for the identification of novel therapeutic targets. However, recognizing the subset of genomic alterations that are functionally relevant has proven difficult. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in a cohort of 222 adult AML patients without known FLT3 mutations and experimentally tested the functional consequences of each candidate leukemogenic allele by exogenous expression in BaF3 cells. DNA sequencing detected nine non-synonymous sequence variants in six exons that were not known single-nucleotide polymorphisms. Functional assessment of these alleles identified four novel activating mutations in the extracellular domain, the juxtamembrane domain, and the activation loop that induced constitutive kinase activity, differentially activated downstream signaling pathways, and conferred varying sensitivity to pharmacologic FLT3 inhibition. In contrast, the remaining five alleles, including mutations in highly conserved, key functional domains of FLT3, were not associated with increased kinase activity and aberrant signal transduction. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation, and underscore the importance of functional validation of candidate alleles discovered using high-throughput genomic screens, to distinguish between ‘driver' mutations underlying cancer development, and biologically neutral ‘passenger' alterations.
PDF
