Peter D. Emanuel

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.

Mutations in CBL occur frequently in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia is an aggressive myeloproliferative disorder characterized by malignant transformation in the hematopoietic stem cell compartment with proliferation of differentiated progeny. Seventy-five percent of patients harbor mutations in the NF1, NRAS, KRAS, or PTPN11 genes, which encode components of Ras signaling networks. Using single nucleotide polymorphism arrays, we identified a region of 11q isodisomy that contains the CBL gene in several JMML samples, and subsequently identified CBL mutations in 27 of 159 JMML samples. Thirteen of these mutations alter codon Y371. In this report, we also demonstrate that CBL and RAS/PTPN11 mutations were mutually exclusive in these patients. Moreover, the exclusivity of CBL mutations with respect to other Ras pathway-associated mutations indicates that CBL may have a role in deregulating this key pathway in JMML.

A randomized trial of dasatinib 100 mg versus imatinib 400 mg in newly diagnosed chronic-phase chronic myeloid leukemia

Tyrosine kinase inhibitor therapy with imatinib (IM), dasatinib (DAS), or nilotinib is very effective in chronic-phase chronic myeloid leukemia. Two hundred fifty-three patients with newly diagnosed chronic-phase chronic myeloid leukemia were randomized to IM 400 mg/day or DAS 100 mg/day. The proportion of patients achieving a complete cytogenetic remission rate was superior with DAS (84% vs 69%), as was the 12-month molecular response by the proportions of patients achieving > 3-log, > 4-log, and > 4.5-log reduction in BCR-ABL transcript levels. Overall and progression-free survival was similar in the 2 arms. Among patients who achieved hematologic CR, 3-year relapse-free survival was 91% with DAS and 88% with IM 400 mg. Grade 3 and 4 toxicities were most commonly hematologic, including thrombocytopenia in 18% and 8% of DAS and IM patients, respectively. DAS induced more complete cytogenetic response and deeper molecular responses after 12 months, compared with IM 400 mg, and with a median follow-up of 3.0 years there have been very few deaths, relapses, or progressions in the 2 arms. In summary, DAS compared with IM appeared to have more short-term cytogenetic and molecular response, more hematologic toxicity, and similar overall survival. This trial is registered at www.clinicaltrials.gov as NCT00070499.

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.

A Pilot Study of Isotretinoin in the Treatment of Juvenile Chronic Myelogenous Leukemia

BACKGROUND Juvenile chronic myelogenous leukemia (CML) is a rare myeloproliferative disease of infants and young children for which there is no effective therapy other than allogeneic bone marrow transplantation. In vitro, isotretinoin (13-cis-retinoic acid) attenuates both the spontaneous proliferation of leukemic peripheral-blood progenitor cells (granulocyte-macrophage colony-forming units) and their selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). We conducted a pilot study to evaluate the clinical efficacy of isotretinoin in juvenile CML. METHODS To be eligible the patients had to have newly diagnosed untreated disease, leukocytosis with monocytosis, marrow with less than 25 percent blasts, hepatosplenomegaly, no chromosomal abnormalities, and negative viral cultures and antibody titers. Isotretinoin was administered orally in single daily doses of 100 mg per square meter of body-surface area. When possible, patients subsequently underwent bone marrow transplantation. RESULTS Ten children (median age, 10 months) were enrolled in the study. In all 10 there was spontaneous colony formation of leukemic progenitor cells in vitro. In the eight patients tested there was hypersensitivity to GM-CSF. The only toxic effect of isotretinoin therapy was cheilitis in two patients. Four children had disease progression. Two children had complete responses to isotretinoin (normalization of the white-cell count and disappearance of organomegaly), three had partial responses (more than a 50 percent reduction in the white-cell count and degree of organomegaly), and one had a minimal response (more than a 50 percent reduction in the white-cell count, but a 26 to 50 percent reduction in the degree of organomegaly). The median duration of response was 37 months (range, 6 to 83). Three of the four children who had a complete or partial response and who did not undergo bone marrow transplantation were alive 36 to 83 months after the diagnosis of juvenile CML. The spontaneous colony formation in vitro was reduced in samples from the five patients in whom this factor was reassessed during treatment. There was also a reduction in the hypersensitivity of leukemic progenitor cells to GM-CSF in the two patients retested. CONCLUSIONS Isotretinoin can induce durable clinical and laboratory responses in patients with juvenile CML.

Juvenile myelomonocytic leukemia: molecular understanding and prospects for therapy

Juvenile myelomonocytic leukemia is a rare but deadly myeloproliferative disorder of early childhood that infrequently progresses to acute leukemia. The pathogenesis of this leukemia has been linked to deregulated signal transduction, resulting in growth factor hypersensitivity. Several other myeloproliferative disorders appear to share growth factor hypersensitivity as a common pathophysiological mechanism and thus this leukemia serves as an important model. New treatment modalities, such as retinoid therapy, are emerging for juvenile myelomonocytic leukemia. Further understanding of deregulated signal transduction should pave the way for even more rationally designed therapy for this leukemia and related disorders.

The genomic landscape of juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm (MPN) of childhood with a poor prognosis. Mutations in NF1, NRAS, KRAS, PTPN11 or CBL occur in 85% of patients, yet there are currently no risk stratification algorithms capable of predicting which patients will be refractory to conventional treatment and could therefore be candidates for experimental therapies. In addition, few molecular pathways aside from the RAS-MAPK pathway have been identified that could serve as the basis for such novel therapeutic strategies. We therefore sought to genomically characterize serial samples from patients at diagnosis through relapse and transformation to acute myeloid leukemia to expand knowledge of the mutational spectrum in JMML. We identified recurrent mutations in genes involved in signal transduction, splicing, Polycomb repressive complex 2 (PRC2) and transcription. Notably, the number of somatic alterations present at diagnosis appears to be the major determinant of outcome.

Myelodysplasia and myeloproliferative disorders in childhood: an update

The myelodysplastic syndromes (MDS) and the myeloproliferative disorders (MPD) arising during childhood, even if combined, are generally thought to represent an uncommon occurrence of a heterogenous group of clonal stem cell disorders. Previous reported incidence rates, based on small series, have suggested that childhood MDS represents approximately 3% of paediatric haematological malignancies. Only two actual population-based studies have been reported for childhood MDS (Hasle et al, 1995; Jackson et al, 1993). The recent study based in Denmark (Hasle et al, 1995), found an annual incidence of 4·0 per million corresponding to 9% of all malignant haematological disorders in children. In contrast, Jackson et al (1993) found a much lower incidence of childhood MDS in the range of 0·5 per million, but there were also significant differences between these studies for the reported incidences of acute myeloid leukaemia, acute lymphoid leukaemia and chronic myeloid leukaemia.

Phase 1-2a multicenter dose-escalation study of ezatiostat hydrochloride liposomes for injection (Telintra®, TLK199), a novel glutathione analog prodrug in patients with myelodysplastic syndrome

BackgroundEzatiostat hydrochloride liposomes for injection, a glutathione S-transferase P1-1 inhibitor, was evaluated in myelodysplastic syndrome (MDS). The objectives were to determine the safety, pharmacokinetics, and hematologic improvement (HI) rate. Phase 1-2a testing of ezatiostat for the treatment of MDS was conducted in a multidose-escalation, multicenter study. Phase 1 patients received ezatiostat at 5 dose levels (50, 100, 200, 400 and 600 mg/m2) intravenously (IV) on days 1 to 5 of a 14-day cycle until MDS progression or unacceptable toxicity. In phase 2, ezatiostat was administered on 2 dose schedules: 600 mg/m2 IV on days 1 to 5 or days 1 to 3 of a 21-day treatment cycle.Results54 patients with histologically confirmed MDS were enrolled. The most common adverse events were grade 1 or 2, respectively, chills (11%, 9%), back pain (15%, 2%), flushing (19%, 0%), nausea (15%, 0%), bone pain (6%, 6%), fatigue (0%, 13%), extremity pain (7%, 4%), dyspnea (9%, 4%), and diarrhea (7%, 4%) related to acute infusional hypersensitivity reactions. The concentration of the primary active metabolites increased proportionate to ezatiostat dosage. Trilineage responses were observed in 4 of 16 patients (25%) with trilineage cytopenia. Hematologic Improvement-Erythroid (HI-E) was observed in 9 of 38 patients (24%), HI-Neutrophil in 11 of 26 patients (42%) and HI-Platelet in 12 of 24 patients (50%). These responses were accompanied by improvement in clinical symptoms and reductions in transfusion requirements. Improvement in bone marrow maturation and cellularity was also observed.ConclusionPhase 2 studies of ezatiostat hydrochloride liposomes for injection in MDS are supported by the tolerability and HI responses observed. An oral formulation of ezatiostat hydrochloride tablets is also in phase 2 clinical development.Trial RegistrationClinicaltrials.gov: NCT00035867

Sickle cell acute chest syndrome associated with parvovirus B19 infection: case series and review.

Acute Chest Syndrome (ACS) continues to be a major source of morbidity and mortality among patients with sickle cell disease. It is characterized by the presence of pleutitic chest pain, fever, rales on lung auscultation, and pulmonary infiltrates on chest X‐ray [Castro et al: Blood 84:643–649, 1994]. The pathophysiology of this disorder remains poorly understood leading to the descriptive term “Acute Chest Syndrome” designated by Charache et al. [Arch Intern Med 139:67–69, 1979]. Typical bacterial pathogens are seldom isolated in adults, although they play a significant role in the pathogenesis of this entity in children. Until recently, the technology to accurately study viral infection as a precipitating cause of ACS has been unavailable.