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Biomedical subjects

Peter D Emanuel

Publications and source records attributed to Peter D Emanuel.

18 recordsLinked to original sources
PubMed2006

Interstitial uniparental isodisomy at clustered breakpoint intervals is a frequent mechanism of NF1 inactivation in myeloid malignancies.

To identify the mechanism of loss of heterozygosity (LOH) and potential modifier gene(s), we investigated the molecular basis of somatic NF1 inactivation in myeloid malignancies from 10 children with neurofibromatosis type 1. Loci across a minimal 50-Mb region of primarily the long arm of chromosome 17 showed LOH in 8 cases, whereas a less than 9-Mb region of loci flanking NF1 had LOH in the remaining 2 cases. Two complementary techniques, quantitative polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), were used to determine whether the copy number at loci that showed LOH was 1 or 2 (ie, deleted or isodisomic). The 2 cases with LOH limited to less than 9 Mb were intrachromosomal deletions. Among the 8 leukemias with 50-Mb LOH segments, 4 had partial uniparental isodisomy and 4 had interstitial uniparental isodisomy. These isodisomic cases showed clustering of the centromeric and telomeric LOH breakpoints. This suggests that the cases with interstitial uniparental isodisomy arose in a leukemia-initiating cell by double-homologous recombination events at intervals of preferred mitotic recombination. Homozygous inactivation of NF1 favored outgrowth of the leukemia-initiating cell. Our studies demonstrate that LOH analyses of loci distributed along the chromosomal length along with copy-number analysis can reveal novel mechanisms of LOH that may potentially identify regions harboring "cryptic" tumor suppressor or modifier genes whose inactivation contributes to tumorigenesis.

Age of Onset↗
PubMed2005

Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease.

Germline mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS) and the clinically related LEOPARD syndrome (LS), whereas somatic mutations in the same gene contribute to leukemogenesis. On the basis of our previously gathered genetic and biochemical data, we proposed a model that splits NS- and leukemia-associated PTPN11 mutations into two major classes of activating lesions with differential perturbing effects on development and hematopoiesis. To test this model, we investigated further the diversity of germline and somatic PTPN11 mutations, delineated the association of those mutations with disease, characterized biochemically a panel of mutant SHP-2 proteins recurring in NS, LS, and leukemia, and performed molecular dynamics simulations to determine the structural effects of selected mutations. Our results document a strict correlation between the identity of the lesion and disease and demonstrate that NS-causative mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated ones. Furthermore, we show that the recurrent LS-causing Y279C and T468M amino acid substitutions engender loss of SHP-2 catalytic activity, identifying a previously unrecognized behavior for this class of missense PTPN11 mutations.

Adult↗
PubMed2005

The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloproliferative disease.

Germ line PTPN11 mutations cause 50% of cases of Noonan syndrome (NS). Somatic mutations in PTPN11 occur in 35% of patients with de novo, nonsyndromic juvenile myelomonocytic leukemia (JMML). Myeloproliferative disorders (MPDs), either transient or more fulminant forms, can also occur in infants with NS (NS/MPD). We identified PTPN11 mutations in blood or bone marrow specimens from 77 newly reported patients with JMML (n = 69) or NS/MPD (n = 8). Together with previous reports, we compared the spectrum of PTPN11 mutations in 3 groups: (1) patients with JMML (n = 107); (2) patients with NS/MPD (n = 19); and (3) patients with NS (n = 243). Glu76 was the most commonly affected residue in JMML (n = 45), with the Glu76Lys alteration (n = 29) being most frequent. Eight of 19 patients with NS/MPD carried the Thr73Ile substitution. These data suggest that there is a genotype/phenotype correlation in the spectrum of PTPN11 mutations found in patients with JMML, NS/MPD, and NS. This supports the need to characterize the spectrum of hematologic abnormalities in individuals with NS and to better define the impact of the PTPN11 lesion on the disease course in patients with NS/MPD and JMML.

Child↗
PubMed2005

The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation.

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5'-untranslated region (5'-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5'-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3'-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5'-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5'-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5'-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5'-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5'-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis.

5' Untranslated Regions↗
PubMed2005

Activating FLT3 mutations are rare in children with juvenile myelomonocytic leukemia.

BACKGROUND: Activating mutations of FLT3 have been identified in multiple myeloid malignancies. Two types of activating mutations have been described: (1) the internal tandem duplication (FLT3-ITD) and (2) point mutations within the activating loop (FLT3-ALM). Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative disorder of early childhood. Mutations and other genetic abnormalities of RAS, NF1, and PTPN11 have been implicated as causative events in JMML, but approximately 25% of JMML patients harbor none of these abnormalities. We investigated whether FLT3 mutations might also contribute to JMML pathogenesis, and if present, whether FLT3 status would correlate with disease natural history and prognosis. PROCEDURES: Genomic DNA was isolated from peripheral blood and bone marrow samples of 60 patients meeting international JMML diagnostic criteria. Samples were analyzed for FLT3-ITD and FLT3-ALM using polymerase chain reaction and restriction endonuclease digestion. RESULTS: FLT3-ALM was found in 1/60 (1.7%) patients analyzed. Direct sequencing confirmed a C836G mutation. Clinical and laboratory characteristics of the JMML patient with the FLT3-ALM did not differ from the remainder of the cohort. No FLT3-ITD mutations were detected. CONCLUSIONS: This first reported mutational analysis for both FLT3-ITD and FLT3-ALM performed in JMML documents the presence of FLT3 mutations within JMML, but at a sufficiently low prevalence as to be clinically insignificant for most patients. Despite the poor prognosis and limited therapeutic options for JMML patients with refractory disease, compassionate therapy with targeted FLT3 inhibitors should not be considered in this patient population until adequate safety and efficacy data become available.

Child↗
PubMed2004

Juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare, clonal, mixed myeloproliferative and myelodysplastic disorder afflicting young children. Patients with JMML respond poorly to most standard chemotherapy regimens and, whereas stem cell transplantation is the only known curative approach, even this modality is hampered by high relapse rates. The pathogenesis of JMML arises from dysregulation of signal transduction through the Ras pathway. This dysregulation results in JMML cells demonstrating selective hypersensitivity in vitro to granulocyte macrophage colony-stimulating factor (GM-CSF). Potential causative mutations or other genetic abnormalities in three genes (eg, RAS, neurofibromatosis type 1, and PTPN11), all of which are positioned in the GM-CSF/Ras signal transduction pathway, account for up to 75% of cases of JMML. These pathogenetic advances are paving the way for the development and testing of mechanism-based molecularly targeted therapeutics in JMML aimed specifically at the GM-CSF signal transduction pathway through Ras.

Algorithms↗
PubMed2003

Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis.

The PTPN11 gene encodes SHP-2 (Src homology 2 domain-containing protein tyrosine Phosphatase), a nonreceptor tyrosine protein tyrosine phosphatase (PTPase) that relays signals from activated growth factor receptors to p21Ras (Ras) and other signaling molecules. Mutations in PTPN11 cause Noonan syndrome (NS), a developmental disorder characterized by cardiac and skeletal defects. NS is also associated with a spectrum of hematologic disorders, including juvenile myelomonocytic leukemia (JMML). To test the hypothesis that PTPN11 mutations might contribute to myeloid leukemogenesis, we screened the entire coding region for mutations in 51 JMML specimens and in selected exons from 60 patients with other myeloid malignancies. Missense mutations in PTPN11 were detected in 16 of 49 JMML specimens from patients without NS, but they were less common in other myeloid malignancies. RAS, NF1, and PTPN11 mutations are largely mutually exclusive in JMML, which suggests that mutant SHP-2 proteins deregulate myeloid growth through Ras. However, although Ba/F3 cells engineered to express leukemia-associated SHP-2 proteins cells showed enhanced growth factor-independent survival, biochemical analysis failed to demonstrate hyperactivation of the Ras effectors extracellular-regulated kinase (ERK) or Akt. We conclude that SHP-2 is an important cellular PTPase that is mutated in myeloid malignancies. Further investigation is required to clarify how these mutant proteins interact with Ras and other effectors to deregulate myeloid growth.

Amino Acid Sequence↗
PubMed2003

Inhibition of tumorigenicity by the 5'-untranslated RNA of the human c-myc P0 transcript.

Activity of the independently regulated human c-myc P0 promoter has been associated with the undifferentiated status of leukemia cells as well as the hormone-independent proliferation of breast cancer cells. The P0 transcript is distinguished from the predominant P1 and P2 c-myc mRNAs by an approximately 639-nucleotide extension of the 5'-untranslated region. We hypothesized that this complex 5'-untranslated RNA sequence unique to the P0 transcript may contribute significantly to the composite regulation of the c-myc locus and that enforced intracellular synthesis of the isolated P0 5'-UTR, out of its native sequence context, might amplify or dominantly interfere with its normal regulatory function. Human tumor (HeLa) cells in which the isolated P0 5'-UTR was ectopically expressed displayed a dramatic decrease in anchorage-independent proliferation. Furthermore, P0 5'-UTR-expressing HeLa cells failed to form tumors when inoculated into SCID mice. This loss of tumorigenicity was associated with increases in levels of the c-Myc1 (p67) and c-Myc2 (p64) proteins and a 3- to 5-fold elevation of spontaneous apoptotic index. These results demonstrate that an isolated 5'-untranslated RNA sequence can be attributed potent in trans gene-regulatory and phenotype-altering capabilities and that extrinsic alterations in c-myc regulation can be utilized to reestablish the natural proapoptotic (tumor suppressor) activities associated with this protooncogene.

5' Untranslated Regions↗
PubMed2003

Evidence for differential ribonucleoprotein complex assembly in vitro on the 5'-untranslated region of the human IGF-IR transcript.

The type I insulin-like growth factor receptor (IGF-IR) plays a key role in the control of cellular proliferation and survival. The human IGF-IR transcript is characterized by an unusually long 1038 nucleotide 5'-untranslated region (5'-UTR). We hypothesized that the contribution of this complex 5'-untranslated RNA sequence to the post-transcriptional regulation of IGF-IR expression would involve a dynamic interplay between RNA structure and specific RNA-binding proteins. Here we have detected and characterized a diverse series of regulatory proteins binding the IGF-IR 5'-UTR under disparate conditions. One pair of proteins ( approximately 42/38 kDa) binds readily to the intact 5'-UTR, which is predicted to adopt a highly base-paired, highly favorable (dG=-498 kcal/mol) three-domain structure. Another protein(s) (p20*) specifically induces formation of a novel RNA structure from within the initial 209 nucleotides of the nascent IGF-IR transcript, but fails to UV crosslink to this RNA sequence. A third group of proteins recognizes and binds the IGF-IR 5'-UTR under highly stringent conditions, but only after higher-ordered RNA structure has been disrupted. Our in vitro results indicate that the IGF-IR 5'-UTR may exist in at least three distinct states, and we propose that interconversion between these states might take place in vivo and differentially alter IGF-IR transcript utilization.

5' Untranslated Regions↗
PubMed2003

The 5'-untranslated RNA of the human dhfr minor transcript alters transcription pre-initiation complex assembly at the major (core) promoter.

The human dhfr minor transcript is distinguished from the predominant dhfr mRNA by an approximately 400 nucleotide extension of the 5'-untranslated region, which corresponds to the major (core) promoter DNA (its template). Based on its unusual sequence composition, we hypothesized that the minor transcript 5'-UTR might be capable of altering transcription pre-initiation complex assembly at the core promoter, through direct interactions of the RNA with specific regulatory polypeptides or the promoter DNA itself. We found that the minor transcript 5'-UTR selectively sequesters transcription factor Sp3, and to a lesser extent Sp1, preventing their binding to the dhfr core promoter. This allows a third putative transcriptional regulatory protein, which is relatively resistant to sequestration by the minor transcript RNA, the opportunity to bind the dhfr core promoter. The selective sequestration of Sp3 > Sp1 by the minor transcript 5'-UTR involves an altered conformation of the RNA, and a structural domain of the protein distinct from that required for binding to DNA. As a consequence, the minor transcript 5'-UTR inhibits transcription from the core promoter in vitro (in trans) in a concentration-dependent manner. These results suggest that the dhfr minor transcript may function in vivo (in cis) to regulate the transcriptional activity of the major (core) promoter.

5' Untranslated Regions↗
PubMed2002

Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis.

A drug that specifically inhibits erythropoiesis would be clinically useful. The erythropoietin (Epo) mutant Epo (R103A) could potentially be used for this purpose. Epo (R103A) has a single amino acid substitution of alanine for arginine at position 103. Because of this mutation, Epo (R103A) is only able to bind to one of the 2 subunits of the erythropoietin receptor (EpoR) homodimer and is thus a competitive inhibitor of Epo activity. To produce large quantities of Epo (R103A) to test in animal models of thalassemia and sickle cell disease, we expressed and purified recombinant Epo (R103A) from the yeast Pichia pastoris. Using this method milligram quantities of highly purified Epo (R103A) are obtained. The yeast-expressed Epo (R103A) is properly processed and glycosylated and specifically inhibits Epo-dependent cell growth and (125)I-Epo binding. Epo (R103A) does not, however, directly induce apoptosis in 32D cells expressing EpoR. Epo (R103A) inhibits erythropoiesis of human CD34(+) hematopoietic cells and completely blocks erythroid burst-forming unit formation in normal human bone marrow colony assays. Yeast-expressed Epo (R103A) is a specific inhibitor of primary erythropoiesis suitable for testing in animal models.

Binding, Competitive↗
PubMed2002

Targeting Raf-1 gene expression by a DNA enzyme inhibits juvenile myelomonocytic leukemia cell growth.

Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood disorder with few therapeutic options. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) promote JMML cell growth. A hyperactive function of the ras oncogene is a hallmark of JMML. We therefore targeted the protein kinase Raf-1 downstream of Ras using a DNA enzyme that degrades mRNA-Raf-1. Western blots of JMML cell lysates revealed phosphorylated Raf-1 protein, indicating constitutive activation. Addition of GM-CSF, but not TNF-alpha, increased phosphorylation of both Raf-1 and the mitogen-activated protein kinases (MAPKs) JNK-1 and ERK-1. Depletion of Raf-1 protein markedly impaired activation of MAPKs, induced substantial inhibition of JMML cell colony formation, and virtually abolished GM-CSF hypersensitivity in JMML cells. Exogenous TNF-alpha, but not GM-CSF, restored colony formation of JMML cells pretreated with the enzyme. We could not detect any effect of the enzyme on the proliferation of normal bone marrow cells, indicating its specificity and potential safety. When immunodeficient mice engrafted with JMML cells were treated continuously with the enzyme via a peritoneal osmotic mini-pump for 4 weeks, a profound reduction in the JMML cell numbers in the recipient murine bone marrows was found. We conclude that GM-CSF is a chief regulator of JMML growth and exerts its proleukemic effects primarily via the Ras/Raf-1 signaling cascade. TNF-alpha plays a permissive role, being dependent upon GM-CSF to induce JMML cell proliferation. The DNA enzyme efficiently catabolized mRNA-Raf-1 with subsequent inhibition of JMML cell growth, suggesting its potential as a mechanism-based therapy in this fatal leukemia.

Bone Marrow Cells↗
PubMed2002

Human herpesvirus 6 infection mimicking juvenile myelomonocytic leukemia in an infant.

In vitro cell culture studies of bone marrow and peripheral blood progenitor cells from patients with juvenile myclomonocytic leukemia (JMML) consistently show spontaneous proliferation and selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). This GM-CSF hypersensitivity dose-response assay has become a component of the international diagnostic criteria for JMML. The authors report a 2-week-old boy with perinatal human herpesvirus 6 (HHV-6) infection in whom in vitro bone marrow culture studies suggested the diagnosis of JMML by showing increased spontaneous proliferation, inhibition of this growth by anti-GM-CSF antibodies, and hypersensitivity to GM-CSF. Polymerase chain reaction viral studies from whole blood DNA and the shell vial viral culture assay were both positive for HHV-6. The patient's condition improved with expectant treatment, with an eventual return to normal blood counts and resolution of hepatosplenomegaly. This case of perinatal HHV-6 infection shows that viruses can initially mimic the in vitro culture results found in patients with JMML. It also illustrates that patients suspected of having JMML should be observed if there are no signs of progressive disease and concurrent features suggestive of viral infection.

Acute Kidney Injury↗
PubMed2002

Atypical cellular disorders.

Atypical cellular disorders are commonly considered part of the gray zone linking oncology to hematology and immunology. Although these disorders are relatively uncommon, they often represent significant clinical problems, provide an opportunity to understand basic disease mechanisms, and serve as model systems for the development of novel targeted therapies. This chapter focuses on such disorders. In Section I, Dr. Arceci discusses the pathogenesis of Langerhans cell histiocytosis (LCH) in terms of the hypothesis that this disorder represents an atypical myeloproliferative syndrome. The clinical manifestations and treatment of LCH in children and adults is discussed along with possible future therapeutic approaches based upon biological considerations. In Section II, Dr. Longley considers the molecular changes in the c-Kit receptor that form the basis of mastocytosis. Based on the location and function of c-Kit mutations, he develops a paradigm for the development of specific, targeted therapies. In Section III, Dr. Emanuel provides a review of the "mixed myeloproliferative and myelodysplastic disorders," including novel therapeutic approaches based on aberrant pathogenetic mechanisms. Taken together, these chapters should provide an overview of the biological basis for these disorders, their clinical manifestations, and new therapeutic approaches.

Adult↗