Alicja M. Gruszka

Overexpression of sPRDM16 coupled with loss of p53 induces myeloid leukemias in mice.

Transgenic expression of the abnormal products of acute myeloid leukemia-associated (AML-associated) primary chromosomal translocations in hematopoietic stem/progenitor cells initiates leukemogenesis in mice, yet additional mutations are needed for leukemia development. We report here aberrant expression of PR domain containing 16 (PRDM16) in AML cells with either translocations of 1p36 or normal karyotype. These carried, respectively, relatively high prevalence of mutations in the TP53 tumor suppressor gene and in the nucleophosmin (NPM) gene, which regulates p53. Two protein isoforms are expressed from PRDM16, which differ in the presence or absence of the PR domain. Overexpression of the short isoform, sPRDM16, in mouse bone marrow induced AML with full penetrance, but only in the absence of p53. The mouse leukemias were characterized by multilineage cellular abnormalities and megakaryocyte dysplasia, a common feature of human AMLs with 1p36 translocations or NPM mutations. Overexpression of sPRDM16 increased the pool of HSCs in vivo, and in vitro blocked myeloid differentiation and prolonged progenitor life span. Loss of p53 augmented the effects of sPRDM16 on stem cell number and induced immortalization of progenitors. Thus, overexpression of sPRDM16 induces abnormal growth of stem cells and progenitors and cooperates with disruption of the p53 pathway in the induction of myeloid leukemia.

A monoclonal antibody against mutated nucleophosmin 1 for the molecular diagnosis of acute myeloid leukemias.

Mutations in the nucleophosmin 1 (NPM1) gene are the most frequent genetic aberrations of acute myeloid leukemia (AML) and define a clinically distinct subset of AML. A monoclonal antibody (T26) was raised against a 19-amino acid polypeptide containing the unique C-terminus of the type A NPM1 mutant protein. T26 recognized 10 of the 21 known NPM1 mutants, including the A, B, and D types, which cover approximately 95% of all cases, and did not cross-react with wild-type NPM1 or unrelated cellular proteins. It performed efficiently with different detection technologies, including immunofluorescence, immunohistochemistry, and flow cytometry. Within a series of consecutive de novo AML patients, 44 of 110 (40%) and 15 of 39 (38%) cases scored positive using the T26 antibody in immunofluorescence and flow cytometry assays, respectively. T26-positive cases were found to be all carrying mutations of NPM1 exclusively, as determined by molecular analysis. T26 is the first antibody that specifically recognizes a leukemia-associated mutant protein. Immunofluorescence or flow cytometry using T26 may thus become a new tool for a rapid, simple, and cost-effective molecular diagnosis of AMLs.

The concurrent use of N- and C-terminal antibodies anti-nucleophosmin 1 in immunofluorescence experiments allows for precise assessment of its subcellular localisation in acute myeloid leukaemia patients.

The concurrent use of N- and C-terminal antibodies anti-nucleophosmin 1 in immunofluorescence experiments allows for precise assessment of its subcellular localisation in acute myeloid leukaemia patients

Recombinant protein quality evaluation: proposal for a minimal information standard

The functionality of the proteins used in biological experiments is very often not assessed at all. Our initiative is aimed at making the scientific community aware about this problem and proposes a first checklist for data reporting. doi:10.4056/sigs.1834511

Nucleophosmin leukemogenic mutant activates Wnt signaling during zebrafish development.

Nucleophosmin (NPM1) is a ubiquitous multifunctional phosphoprotein with both oncogenic and tumor suppressor functions. Mutations of the NPM1 gene are the most frequent genetic alterations in acute myeloid leukemia (AML) and result in the expression of a mutant protein with aberrant cytoplasmic localization, NPMc+. Although NPMc+ causes myeloproliferation and AML in animal models, its mechanism of action remains largely unknown. Here we report that NPMc+ activates canonical Wnt signaling during the early phases of zebrafish development and determines a Wnt-dependent increase in the number of progenitor cells during primitive hematopoiesis. Coherently, the canonical Wnt pathway is active in AML blasts bearing NPMc+ and depletion of the mutant protein in the patient derived OCI-AML3 cell line leads to a decrease in the levels of active β-catenin and of Wnt target genes. Our results reveal a novel function of NPMc+ and provide insight into the molecular pathogenesis of AML bearing NPM1 mutations.

PML-RARA-associated cooperating mutations belong to a transcriptional network that is deregulated in myeloid leukemias

It has been shown that individual acute myeloid leukemia (AML) patients are characterized by one of few initiating DNA mutations and 5–10 cooperating mutations not yet defined among hundreds identified by massive sequencing of AML genomes. We report an in vivo insertional-mutagenesis screen for genes cooperating with one AML initiating mutations (PML-RARA, oncogene of acute promyelocytic leukemia, APL), which allowed identification of hundreds of genetic cooperators. The cooperators are mutated at low frequency in APL or AML patients but are always abnormally expressed in a cohort of 182 APLs and AMLs analyzed. These deregulations appear non-randomly distributed and present in all samples, regardless of their associated genomic mutations. Reverse-engineering approaches showed that these cooperators belong to a single transcriptional gene network, enriched in genes mutated in AMLs, where perturbation of single genes modifies expression of others. Their gene-ontology analysis showed enrichment of genes directly involved in cell proliferation control. Therefore, the pool of PML-RARA cooperating mutations appears large and heterogeneous, but functionally equivalent and deregulated in the majority of APLs and AMLs. Our data suggest that the high heterogeneity of DNA mutations in APLs and AMLs can be reduced to patterns of gene expression deregulation of a single ‘mutated’ gene network.

AML1/ETO accelerates cell migration and impairs cell-to-cell adhesion and homing of hematopoietic stem/progenitor cells

The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression.

Understanding the molecular basis of acute myeloid leukemias: where are we now?

Although the treatment modalities for acute myeloid leukemia (AML) have not changed much over the past 40 years, distinct progress has been made in deciphering the basic biology underlying the pathogenesis of this group of hematological disorders. Studies show that AML development is a multicause, multistep and multipathway process. Accordingly, AMLs constitute a heterogeneous group of diseases. The thorough understanding of the molecular basis of AML is paving the way for better therapeutic approaches. Multiple novel drugs are being introduced and new, more efficient and less toxic formulations of conventional therapeutics are becoming available. Here, we review the recent advances in the comprehension of the molecular processes that lead to the onset of AML and its translation into clinical practice.

Molecular investigation of coexistent chronic myeloid leukaemia and peripheral T-cell lymphoma-a case report

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm underlain by the formation of BCR-ABL1 – an aberrant tyrosine kinase – in the leukaemic blasts. Long-term survival rates in CML prior to the advent of tyrosine kinase inhibitors (TKIs) were dismal, albeit the incidence of secondary malignancies was higher than that of age-matched population. Current figures confirm the safety of TKIs with conflicting data concerning the increased risk of secondary tumours. We postulate that care has to be taken when distinguishing between coexisting, secondary-to-treatment and second in sequence, but independent tumourigenic events, in order to achieve an unbiased picture of the adverse effects of novel treatments. To illustrate this point, we present a case of a patient in which CML and peripheral T-cell lymphoma (PTCL) coexisted, although the clinical presentation of the latter followed the achievement of major molecular response of CML to TKIs.

PML/RARα fusion gene and response to retinoic acid and arsenic trioxide treatment

Chapter 14 MammaPrint for Individualized Recurrence Risk Assessment and Treatment Recommendations for Early-Stage Breast Cancer Patients Sonal J. Desai and Tianhong Li University of California Davis Comprehensive Cancer Center, Division of Hematology & Oncology, Sacramento, CA 95817, USA sonal.desai@ucdmc.ucdavis.edu, tianhong.li@ucdmc.ucdavis.edu 14.1 Introduction Breast cancer is the second most common cancer and the second leading cause of cancer-related deaths in women worldwide. In 2013 an estimated 232,340 women are expected to be diagnosed with new cases of invasive breast cancers in the United States, and 39,620 women are expected to die from the disease [1, 2]. The majority of these deaths are due to disease recurrence or distant metastasis after initial treatment. Adjuvant systemic therapy with either endocrine therapy and/or chemotherapy has been shown to reduce the risk of distant recurrence and death from invasive breast cancer after local treatment with surgery with or without radiation therapy. To save lives, existing guidelines, aimed at avoiding under use of adjuvant Handbook of Therapeutic Biomarkers in Cancer Edited by Sherry X. Yang and Janet E. Dancey Copyright