Structural insights into amyloid structures of the C-terminal region of nucleophosmin 1 in type A mutation of acute myeloid leukemia

Acute myeloid leukemia (AML) is a clinically and a molecularly heterogeneous disease characterized by the accumulation of undifferentiated and uncontrolled proliferation of hematopoietic progenitor cells. The sub-group named “AML with gene mutations” includes mutations in nucleophosmin (NPM1) assumed as a distinct leukemic entity. NPM1 is an abundant multifunctional protein belonging to the nucleoplasmin family of nuclear chaperones. AML mutated protein is translocated into the cytoplasm (NPM1c+) retaining all functional domains except the loss of a unique NoLs (nucleolar localization signal) at the C-term domain (CTD) and the subsequent disruption of a three helix bundle as tertiary structure. The oligomeric state of NPM1 is of outmost importance for its biological roles and our previous studies linked an aggregation propensity of distinct regions of CTD to leukomogenic potentials of AML mutations. Here we investigated a polypeptide spanning the third and second helices of the bundle of type A mutated CTD. By a combination of several techniques, we ascertained the amyloid character of the aggregates and of fibrils resulting from a self-recognition mechanism. Further amyloid assemblies resulted cytoxic in MTT assay strengthening a new idea of a therapeutic strategy in AML consisting in the self-degradation of mutated NPM1.     

Type C mutation of nucleophosmin 1 acute myeloid leukemia: Consequences of intrinsic disorder

BackgroundNucleophosmin 1 (NPM1) protein is a multifunctional nucleolar chaperone and its gene is the most frequently mutated in Acute Myeloid Leukemia (AML). AML mutations cause the unfolding of the C-terminal domain (CTD) and the protein delocalizing in the cytosol (NPM1c+). Marked aggregation endowed with an amyloid character was assessed as consequences of mutations.ScopeHerein we analyzed the effects of type C mutation on two protein regions: i) a N-terminal extended version of the CTD, named Cterm_mutC and ii) a shorter polypeptide including the sequences of the second and third helices of the CTD, named H2_mutC.Major conclusionsBoth demonstrated able to self-assembly with different kinetics and conformational intermediates and to provide fibers presenting large flexible regions.General significanceThe present study adds a new piece of knowledge to the effects of AML-mutations on structural biology of Nucleophosmin 1, that could be exploited in therapeutic interventions targeting selectively NPMc+.     

The origin and evolution of mutations in acute myeloid leukemia

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.     

The frequency of NPM1 mutations in childhood acute myeloid leukemia

Leser-Trélat syndrome is characterized by the eruptive appearance of multiple seborrheic keratoses in association with underlying malignant disease. Usually, the sign of Leser-Trélat is associated with adenocarcinoma, most frequently of the colon, breast, or stomach, but also of the lung, kidney, liver, and pancreas. Herein, we present a case that we believe is the first report of the sign of Leser-Trélat in association with occult gastric adenocarcinoma and the anamnestic oncologic history of five other multiple primitive cancers. Epidermal growth factor receptor (EGFR) immunohistochemical expression analysis of multiple seborrheic keratoses revealed an intense membranous staining in the basal keratinocytes and in all the upper epidermal layers. Patients with the sign of Leser-Trélat should undergo a diagnostic screening programme for malignant disease along with patients with known Leser-Trélat syndrome who present with a recent acute and florid eruption of their seborrheic keratoses. We propose the importance of combining the molecular features of multiple seborrheic keratoses with EGFR immunohistochemistry analyses to determine the likelihood of Leser-Trélat syndrome and the consequent high risk of underlying multiple visceral malignancies.     

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

Nucleophosmin (NPM1) is an abundant, ubiquitously expressed protein mainly localized at nucleoli but continuously shuttling between nucleus and cytoplasm. NPM1 plays a role in several cellular functions, including ribosome biogenesis and export, centrosome duplication, chromatin remodeling, DNA repair, and response to stress stimuli. Much of the interest in this protein arises from its relevance in human malignancies. NPM1 is frequently overexpressed in solid tumors and is the target of several chromosomal translocations in hematologic neoplasms. Notably, NPM1 has been characterized as the most frequently mutated gene in acute myeloid leukemia (AML). Mutations alter the C‐terminal DNA‐binding domain of the protein and result in its aberrant nuclear export and stable cytosolic localization. In this review, we focus on the leukemia‐associated NPM1 C‐terminal domain and describe its structure, function, and the effect exerted by leukemic mutations. Finally, we discuss the possibility to target NPM1 for the treatment of cancer and, in particular, of AML patients with mutated NPM1 gene.     

Annular promyelocytes in acute myeloid leukemia

Granulocytes with ring-shaped nuclei (annular granulocytes; ring granulocytes) are normal bone marrow constituents in rodents. Studies in man have shown a small number of these cells in cases of myeloproliferative diseases. Myelocytes and metamyelocytes have also been described. Similar to rodents and some other animal species, the annular promyelocyte also exists in humans. The significance of these very rare cells in human haemopoiesis becomes an interesting question.     

Interleukin-17 in acute myeloid leukemia

There are several reports that angiogenesis plays important roles in hematological malignancies including acute myeloid leukemia (AML). Human interleukin-17 (IL-17) is a proinflammatory cytokine produced by activated CD4 T cells. IL-17 plays a potential role in T cell mediated angiogenesis. The role of IL-17 in pathologic angiogenesis has not been evaluated yet. The aim of the study was to determine plasma level of IL-17 in patients with AML. IL-17 levels were measured by ELISA in plasma samples taken from 68 adult patients with AML before chemotherapy was administered. In addition 20 out of 68 patients were reanalysed after achieving complete remission (CR). Ten samples from healthy volunteers were evaluated as the control. In this study we have demonstrated that serum level of IL-17 is not elevated in AML patients. These results suggest that angiogenesis in AML is not mediated by CD4 T cells. To our knowledge this is the first report about IL-17 serum level in acute leukemias. We are currently evaluating IL-17 levels in others haematological malignancies.     

Differential signaling of Flt3 activating mutations in acute myeloid leukemia: a working model

Receptor tyrosine kinases couple a wide variety of extracellular cues to cellular responses. The class III subfamily comprises the platelet-derived growth factor receptor, c-Kit, Flt3 and c-Fms, all of which relay cell proliferation signals upon ligand binding. Accordingly, mutations in these proteins that confer ligand-independent activation are found in a subset of cancers. These mutations cluster in the juxtamembrane (JM) and catalytic tyrosine kinase domain (TKD) regions. In the case of acute myeloid leukemia (AML), the juxtamembrane (named ITD for internal tandem duplication) and TKD Flt3 mutants differ in their spectra of clinical outcomes. Although the mechanism of aberrant activation has been largely elucidated by biochemical and structural analyses of mutant kinases, the differences in disease presentation cannot be attributed to a change in substrate specificity or signaling strength of the catalytic domain. This review discusses the latest literature and presents a working model of differential Flt3 signaling based on mis-localized juxtamembrane autophosphorylation, to account for the disease variation. This will have bearing on therapeutic approaches in a complex disease such as AML, for which no efficacious drug yet exists.     

Genetic profile of acute myeloid leukemia

Understanding genomic events and the cascade of their effects in cell function is crucial for identifying distinct subsets of acute myeloid leukemia and developing new therapeutic strategies. Conventional cytogenetics, fluorescence in situ hybridization investigations and molecular studies have provided much information over the past few years. This review will focus on major genomic mechanisms in acute myeloid luekemia and on the genes implicated in the pathogenesis of specific subtypes.     

Antiapoptotic microenvironment of acute myeloid leukemia

We showed previously that tumor-derived supernatant (TSN) from acute myeloid leukemia (AML) myeloblasts inhibits peripheral blood T cell activation and proliferation, rendering the T cells functionally incompetent. We show here that the AML TSN also significantly delays apoptosis of both resting and stimulated T cells, as judged by reduction in annexin V/propidium iodide staining. In addition, we show that this is not unique to T cells and that AML TSN inhibits apoptosis of peripheral B cells, neutrophils, and monocytes. Furthermore, it also enhances the survival of other AML myeloblasts with lower viability. Investigations into the mechanism demonstrate a reduction in the cleavage of procaspase-3, -8, and -9 and the caspase substrate, poly(ADP-ribose)polymerase (PARP). This may be due to Bcl-2, which is normally down-regulated in CD3/CD28-stimulated T cells, but is maintained in the presence of AML TSN. We conclude that AML cells generate an antiapoptotic microenvironment that favors the survival of malignant cells, but also inhibits apoptosis of other normal hemopoietic cells. Reversal of these immunosuppressive effects and restoration of normal immune responses in patients with AML would improve the success of immunotherapy protocols.     

FLT3 inhibitors in acute myeloid leukemia

FLT3 mutations are one of the most common findings in acute myeloid leukemia (AML). FLT3 inhibitors have been in active clinical development. Midostaurin as the first-in-class FLT3 inhibitor has been approved for treatment of patients with FLT3-mutated AML. In this review, we summarized the preclinical and clinical studies on new FLT3 inhibitors, including sorafenib, lestaurtinib, sunitinib, tandutinib, quizartinib, midostaurin, gilteritinib, crenolanib, cabozantinib, Sel24-B489, G-749, AMG 925, TTT-3002, and FF-10101. New generation FLT3 inhibitors and combination therapies may overcome resistance to first-generation agents.     

Dendritic cell vaccination in acute myeloid leukemia

The prognosis of patients with acute myeloid leukemia (AML) remains dismal, with a 5-year overall survival rate of only 5.2% for the continuously growing subgroup of AML patients older than 65 years. These patients are generally not considered eligible for intensive chemotherapy and/or allogeneic hematopoietic stem cell transplantation because of high treatment-related morbidity and mortality, emphasizing the need for novel, less toxic, treatment alternatives. It is within this context that immunotherapy has gained attention in recent years. In this review, we focus on the use of dendritic cell (DC) vaccines for immunotherapy of AML. DC are central orchestrators of the immune system, bridging innate and adaptive immunity and critical to the induction of anti-leukemic immunity. We discuss the rationale and basic principles of DC-based therapy for AML and review the clinical experience that has been obtained so far with this form of immunotherapy for patients with AML.     

Modifying the biological response in acute myeloid leukemia

Summary Lymphocyte response to mitogens and to lymphocyte suppressor and monocyte helper activity was studied in 18 patients with acute myeloid leukemia in complete remission, and in 17 healthy controls. Ten patients were maintained with chemotherapy alone (CT), and eight received chemoimmunotherapy with BCG + leukemic cells (CIT).In late remission the mitogen responsiveness was increased in CT patients and decreased in CIT patients.No significant difference in lymphocyte suppressor activity could be demonstrated between patients and controls, or between CT and CIT.When autologous CIT monocytes were added to mitogen-stimulated lymphocytes they acted as helper cells. CT monocytes, in contrast, seemed to act as suppressor cells. Control monocytes also acted as helper cells, but to a significantly lesser degree than CIT monocytes.Fellow, Hospital Universitario de Caracas, Division of Hematology, Universidad Central de Venezuela, Caracas, VenezuelaCentral Microbiological Laboratory of Stockholm Country CouncilAnna Villa Rusconi fellow, Department of Internal Medicine, University of Ferrara, Italy