Claudia Voena

The anaplastic lymphoma kinase in the pathogenesis of cancer

Tyrosine kinases are involved in the pathogenesis of most cancers. However, few tyrosine kinases have been shown to have a well-defined pathogenetic role in lymphomas. The anaplastic lymphoma kinase (ALK) is the oncogene of most anaplastic large cell lymphomas (ALCL), driving transformation through many molecular mechanisms. In this Review, we will analyse how translocations or deregulated expression of ALK contribute to oncogenesis and how recent genetic or pharmacological tools, aimed at neutralizing its activity, can represent the basis for the design of powerful combination therapies.

Molecular and Clinical Remissions in Multiple Myeloma: Role of Autologous and Allogeneic Transplantation of Hematopoietic Cells

PURPOSE To describe molecular monitoring of minimal residual disease in patients with myeloma who have achieved complete remission (CR) after autologous or allogeneic transplantation of hematopoietic cells. MATERIALS AND METHODS Clonal markers based upon the rearrangement of immunoglobulin heavy-chain genes were generated for each patient and used for polymerase chain reaction (PCR) detection of residual myeloma cells. Fifty-one patients entered the program and 36 achieved CR. After transplantation, molecular monitoring was performed on 29 patients (15 autologous and 14 allogeneic transplants) who had molecular markers. RESULTS Our data show that molecular remissions are rarely achieved (7%) with high-dose chemotherapy followed by single or double autografting. In addition, virtually all peripheral blood progenitor cell and bone marrow samples contained residual myeloma cells, even when sample collection was scheduled after repeated courses of high-dose chemotherapy. All patients autografted with PCR-positive cells remain positive, and eight of 15 have relapsed. Two patients were autografted with PCR-negative cells: one is in clinical and molecular remission, and one relapsed 25 months after the transplant. In the allografting setting, a higher proportion of patients (50%) achieved molecular remission; there were two relapses, one in the PCR-positive group and one in the PCR-negative group. CONCLUSION This is the first large study of molecular remissions in myeloma patients to use a PCR-based approach utilizing patient-specific tumor markers. The sizeable fraction of patients who achieved molecular remission after allografting with peripheral blood progenitor cells represents a promising finding in an incurable disease.

Simple and Rapid In Vivo Generation of Chromosomal Rearrangements using CRISPR/Cas9 Technology

Generation of genetically engineered mouse models (GEMMs) for chromosomal translocations in the endogenous loci by a knockin strategy is lengthy and costly. The CRISPR/Cas9 system provides an innovative and flexible approach for genome engineering of genomic loci in vitro and in vivo. Here, we report the use of the CRISPR/Cas9 system for engineering a specific chromosomal translocation in adult mice in vivo. We designed CRISPR/Cas9 lentiviral vectors to induce cleavage of the murine endogenous Eml4 and Alk loci in order to generate the Eml4-Alk gene rearrangement recurrently found in non-small-cell lung cancers (NSCLCs). Intratracheal or intrapulmonary inoculation of lentiviruses induced Eml4-Alk gene rearrangement in lung cells in vivo. Genomic and mRNA sequencing confirmed the genome editing and the production of the Eml4-Alk fusion transcript. All mice developed Eml4-Alk-rearranged lung tumors 2 months after the inoculation, demonstrating that the CRISPR/Cas9 system is a feasible and simple method for the generation of chromosomal rearrangements in vivo.

A novel nested-PCR strategy for the detection of rearranged immunoglobulin heavy-chain genes in B cell tumors.

Several methods have been developed for the detection of minimal residual disease (MRD) in B cell tumors. Chromosomal translocations or the rearrangement of the immunoglobulin heavy chain (IgH) and T cell receptor genes are generally employed. We report a novel PCR method to detect MRD using IgH genes. IgH rearranged variable region (VDJ) were amplified from tumor specimens using consensus primers for variable and joining region genes. Complementarity-determining regions (CDR) were identified and used to generate tumor-specific primers. Two-round amplifications using primers derived from CDRs and joining or constant regions were performed for MRD detection. IgH nested-PCR approach was tested on a panel of 75 B cell tumors including acute lymphoblastic and chronic lymphocytic leukemias, non-Hodgkin’s lymphomas and multiple myelomas. A VDJ sequence was obtained in 62 out of 75 cases (83%). Sensitivity using DNA or cDNA templates was 10−5 and −6, respectively. This method is specific and sensitive and provides a simple, non-radioactive approach for the evaluation of MRD in B cell tumors.

The Tyrosine Phosphatase Shp2 Interacts with NPM-ALK and Regulates Anaplastic Lymphoma Cell Growth and Migration

Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration.

Maspin and mammaglobin genes are specific markers for RT-PCR detection of minimal residual disease in patients with breast cancer

BACKGROUND This study evaluates the specificity of some reverse-transcriptase polymerase chain reaction (RT-PCR) assays for the detection of residual tumor cells in breast cancer patients. The following markers have been analysed: carcinoembryonic antigen (CEA), cytokeratins (CK19 and CK20), polymorphic epithelial mucin (MUC-1), epidermal growth factor receptor (EGFR), maspin, and mammaglobin. RT-PCR was employed to detect breast cancer cells in peripheral blood (PB), bone marrow (BM), and stem cell leukoaphereses (PBPC). PATIENTS AND METHODS We evaluated the specificity of our RT-PCR assays on a panel of breast cancer specimens (n = 30), on PBPC in patients undergoing high-dose chemotherapy (n = 38), on BM (n = 7) and PB (n = 5) samples obtained from patients with breast cancer. Marrow cells, PB, and PBPC from normal subjects or hematological tumor patients were tested as negative controls. RESULTS Only maspin and mammaglobin met the criteria of sensitivity and specificity required for the detection of residual disease; they were expressed in 80% and 97% of breast cancer specimens, respectively, and not expressed in normal controls. CK19, CK20. EGFR, MUC-1, and CEA were sometimes expressed in normal blood cells and/or hematological tumors. CONCLUSIONS Our data support the notion that maspin and mammaglobin are useful markers for RT-PCR detection of minimal residual disease (MRD) in breast cancer patients, and that perspective clinical studies are needed to determine wether RT-PCR assays will be useful in assessing prognosis, tailoring therapy, or developing new strategies for ex vivo purging.

NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell-specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell-specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor-related signaling molecules, including CD3epsilon, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK(K210R), downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling.

PCR-Detectable Nonneoplastic Bcl-2/IgH Rearrangements Are Common in Normal Subjects and Cancer Patients at Diagnosis but Rare in Subjects Treated With Chemotherapy

PURPOSE To assess whether nonneoplastic Bcl-2/IgH rearrangements act as a confounding factor in the setting of minimal residual disease analysis by evaluating their incidence in a panel of lymphoma-free subjects, including cancer-free donors and chemotherapy-naive and chemotherapy-treated cancer patients. PATIENTS AND METHODS A total of 501 nonlymphoma subjects have been assessed: 258 cancer-free patients and 243 patients with malignancies other than lymphoma, 112 of whom were chemotherapy-naive. Patients were primarily assessed by nested polymerase chain reaction (PCR), followed by real-time quantitative PCR if they scored positive. In addition, six initially PCR-positive cancer-free donors were prospectively reassessed by qualitative and quantitative PCR after 30 and 60 days. RESULTS The overall incidence of Bcl-2/IgH positivity was 9.6%, with a median number of 11 rearrangements per 1,000,000 diploid genomes (range, 0 to 2,845 rearrangements), as assessed by real-time PCR. The incidence was similar in healthy subjects and cancer patients at diagnosis (12% and 12.5%; P = not significant). In contrast, the incidence of this translocation was only 2.3% in chemotherapy-treated patients (P <.001). In addition, three initially PCR-positive cancer-free donors showed persistence of their rearrangements when assessed after 30 and 60 days. CONCLUSION The low incidence of nonneoplastic Bcl-2/IgH rearrangements following chemotherapy provides further evidence of the prognostic role of persistent PCR-positivity in the posttreatment molecular follow-up of follicular lymphoma patients.

The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL

Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

The Anaplastic Lymphoma Kinase Controls Cell Shape and Growth of Anaplastic Large Cell Lymphoma through Cdc42 Activation

Anaplastic large cell lymphoma (ALCL) is a non-Hodgkins lymphoma that originates from T cells and frequently expresses oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. The proliferation and survival of ALCL cells are determined by the ALK activity. Here we show that the kinase activity of the nucleophosmin (NPM)-ALK fusion regulated the shape of ALCL cells and F-actin filament assembly in a pattern similar to T-cell receptor-stimulated cells. NPM-ALK formed a complex with the guanine exchange factor VAV1, enhancing its activation through phosphorylation. VAV1 increased Cdc42 activity, and in turn, Cdc42 regulated the shape and migration of ALCL cells. In vitro knockdown of VAV1 or Cdc42 by short hairpin RNA, as well as pharmacologic inhibition of Cdc42 activity by secramine, resulted in a cell cycle arrest and apoptosis of ALCL cells. Importantly, the concomitant inhibition of Cdc42 and NPM-ALK kinase acted synergistically to induce apoptosis of ALCL cells. Finally, Cdc42 was necessary for the growth as well as for the maintenance of already established lymphomas in vivo. Thus, our data open perspectives for new therapeutic strategies by revealing a mechanism of regulation of ALCL cell growth through Cdc42.