Frederick Racke

Activation of Transferrin Receptor 1 by c-Myc Enhances Cellular Proliferation and Tumorigenesis

ABSTRACT Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.

Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model

Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) mutations is one of the most common molecular alterations known in acute myeloid leukemia (AML). To investigate the role FLT3/ITD mutations play in the development of leukemia, we generated a FLT3/ITD knock-in mouse model by inserting an ITD mutation into the juxtamembrane domain of murine Flt3. FLT3wt/ITD mice developed myeloproliferative disease, characterized by splenomegaly, leukocytosis, and myeloid hypercellularity, which progressed to mortality by 6 to 20 months. Bone marrow (BM) and spleen from FLT3wt/ITD mice had an increased fraction of granulocytes/monocytes and dendritic cells, and a decreased fraction of B-lymphocytes. No sign of acute leukemia was observed over the lifetime of these mice. BM from FLT3wt/ITD mice showed enhanced potential to generate myeloid colonies in vitro. BM from FLT3wt/ITD mice also produced more spleen colonies in the in vivo colony-forming unit (CFU)-spleen assay. In the long-term competitive repopulation assay, BM cells from FLT3wt/ITD mice outgrew the wild-type competitor cells and showed increased myeloid and reduced lymphoid expansion activity. In summary, our data indicate that expression of FLT3/ITD mutations alone is capable of conferring normal hematopoietic stem/progenitor cells (HSPCs) with enhanced myeloid expansion. It also appears to suppress B lymphoid maturation. Additional cooperative events appear to be required to progress to acute leukemia.

Aberrant Overexpression of IL-15 Initiates Large Granular Lymphocyte Leukemia through Chromosomal Instability and DNA Hypermethylation

How inflammation causes cancer is unclear. Interleukin-15 (IL-15) is a pro-inflammatory cytokine elevated in human large granular lymphocyte (LGL) leukemia. Mice overexpressing IL-15 develop LGL leukemia. Here, we show that prolonged in vitro exposure of wild-type (WT) LGL to IL-15 results in Myc-mediated upregulation of aurora kinases, centrosome aberrancies, and aneuploidy. Simultaneously, IL-15 represses miR-29b via induction of Myc/NF-κBp65/Hdac-1, resulting in Dnmt3b overexpression and DNA hypermethylation. All this is validated in human LGL leukemia. Adoptive transfer of WT LGL cultured with IL-15 led to malignant transformation in vivo. Drug targeting that reverses miR-29b repression cures otherwise fatal LGL leukemia. We show how excessive IL-15 initiates cancer and demonstrate effective drug targeting for potential therapy of human LGL leukemia.

CD1d on Myeloid Dendritic Cells Stimulates Cytokine Secretion from and Cytolytic Activity of Vα24JαQ T Cells: A Feedback Mechanism for Immune Regulation

The precise immunologic functions of CD1d-restricted, CD161+ AV24AJ18 (Vα24JαQ) T cells are not well defined, although production of IL-4 has been suggested as important for priming Th2 responses. However, activation of human Vα24JαQ T cell clones by anti-CD3 resulted in the secretion of multiple cytokines notably important for the recruitment and differentiation of myeloid dendritic cells. Specific activation of Vα24JαQ T cells was CD1d restricted. Expression of CD1d was found on monocyte-derived dendritic cells in vitro, and immunohistochemical staining directly revealed CD1d preferentially expressed on dendritic cells in the paracortical T cell zones of lymph nodes. Moreover, myeloid dendritic cells both activated Vα24JαQ T cells and were susceptible to lysis by these same regulatory T cells. Because myeloid dendritic cells are a major source of IL-12 and control Th1 cell differentiation, their elimination by lysis is a mechanism for limiting the generation of Th1 cells and thus regulating Th1/Th2 responses.

Jun Blockade of Erythropoiesis: Role for Repression of GATA-1 by HERP2

ABSTRACT Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation.

Pseudo-spikes are common in histologically benign lymphoid tissues.

T cell receptor gene rearrangement is a classic marker of T cell clonality and is a useful adjunct in the diagnosis of T cell lymphomas and leukemias. Rearranged V-J gene segments amplified by polymerase chain reaction (PCR) are traditionally analyzed by polyacrylamide gel electrophoresis. We and others have analyzed TCR-gamma PCR products using capillary gel electrophoresis, which produces single nucleotide resolution and provides improved diagnostic sensitivity over conventional methods. However, with this marked increase in resolution and sensitivity, it is necessary to re-define normal variation of TCR-gamma gene rearrangement in control tissues to allow appropriate interpretation of monoclonality if present. Using DNA capillary gel electrophoresis, we examined the spectrum of normal patterns for TCR-gamma in a variety of T-cell-rich, histologically benign tissue types, including spleen, lymph node, tonsil, and blood, and compared this with the patterns in T cell lymphoma samples. We defined relative peak heights as h1/h2, where h1 represents the peak height of the largest peak above the normally distributed population, and h2 represents the peak height of the normally distributed curve. We found spikes in almost 20% of histologically benign samples with relative peak heights that were more than 0.5 and up to 1.5. We designated these as pseudo-spikes, because they may be mistaken for monoclonal spikes. In contrast, the relative peak height of the T cell lymphoma samples that showed clonal rearrangement was much higher than that of the pseudo-spikes, being at least 2 in 11/11 and at least 3 in 10/11 cases. Our data suggest that peaks with relative height of at least 3 represent a true clonal population in diagnostic samples. Peaks with relative heights of less than 1.5 may be insignificant, while peaks with relative heights between 1.5 to 3 may warrant further evaluation. Although capillary gel electrophoresis is superior in assessing T cell clonality, caution must be exercised when interpreting results, because pseudo-spikes appear to be common in benign tissues with lymphoid populations and are not necessarily indicative of clonal malignant T cell population.

Defective Human MutY Phosphorylation Exists in Colorectal Cancer Cell Lines with Wild-type MutY Alleles

Oxidative DNA damage can generate a variety of cytotoxic DNA lesions such as 8-oxoguanine (8-oxoG), which is one of the most mutagenic bases formed from oxidation of genomic DNA because 8-oxoG can readily mispair with either cytosine or adenine. If unrepaired, further replication of A·8-oxoG mispairs results in C:G to A:T transversions, a form of genomic instability. We reported previously that repair of A·8-oxoG mispairs was defective and that 8-oxoG levels were elevated in several microsatellite stable human colorectal cancer cell lines lacking MutY mutations (human MutY homolog gene, hmyh, MYH MutY homolog protein). In this report, we provide biochemical evidence that the defective repair of A·8-oxoG may be due, at least in part, to defective phosphorylation of the MutY protein in these cell lines. In MutY-defective cell extracts, but not extracts with functional MutY, A·8-oxoG repair was increased by incubation with protein kinases A and C (PKA and PKC) and caesin kinase II. Treatment of these defective cells, but not cells with functional MutY, with phorbol-12-myristate-13-acetate also increased the cellular A·8-oxoG repair activity and decreased the elevated 8-oxoG levels. We show that MutY is serine-phosphorylated in vitro by the action of PKC and in the MutY-defective cells by phorbol-12-myristate-13-acetate but that MutY is already phosphorylated at baseline in proficient cell lines. Finally, using antibody-isolated MutY protein, we show that MutY can be directly phosphorylated by PKC that directly increases the level of MutY catalyzed A·8-oxoG repair.

Mantle Cell Lymphoma 12 Years After Allogeneic Bone Marrow Transplantation Occurring Simultaneously in Recipient and Donor

A 34-year-old woman was diagnosed with chronic myeloid leukemia in chronic phase in October 1995 after she was found to have an elevated WBC count on routine evaluation. A six of six HLA-matched, sex-mismatched, ABO-mismatched, unrelated donor was identified from the National Marrow Donor Program pool, and in June 1996, the patient underwent a bone marrow transplantation. Her post-transplantation course was complicated by chronic graftversus-host disease. By December 2000, her immunosuppression for graft-versus-host disease was tapered off completely, and her chronic myeloid leukemia remained in morphologic, cytogenetic, and molecular remission. In April 2008, the patient developed cervical and inguinal lymphadenopathy. She underwent a biopsy of a right inguinal lymph node on May 20, 2008, which demonstrated mantle cell lymphoma (MCL) with immunohistochemistry confirming expression of nuclear cyclin D1. She had stage IVA disease with lymphadenopathy, a WBC of 5,200/ L, an absolute lymphocyte count of 900/ L, 5% peripheral blood involvement by flow cytometry, and bone marrow with 1.7% involvement by flow cytometry. Fluorescent in situ hybridization (FISH) analysis of the tumor cells in the lymph node specimen demonstrated that the malignant cells contained the Y chromosome, strongly suggesting that the MCL was donor derived. Figure 1A shows recipient MCL cells at the capsular region, with some of the lymphoma cells infiltrating into the capsules (hematoxylin and eosin H&E, 400). FISH (Fig 1B) highlights the lymphoma cells with XY chromosomes (one orange and one green) in the lymph node and infiltrating the capsule (white arrows), whereas host stroma cells have XX (double green) chromosomes (gold arrows, 1,000). FISH analysis of recipient peripheral blood mononuclear cells for the XY signal revealed 100% donor hematopoietic chimerism at the time of the patient’s MCL diagnosis. This prompted further evaluation of the origin of the MCL. The patient’s stem cell donor is a 61-year-old man who was diagnosed with MCL on May 5, 2008, during the same month as the recipient. He presented with dysphagia and weight loss and was found to have a 3.3-cm tonsillar mass. Biopsy of the mass demonstrated MCL cells (Fig 1C; hematoxylin and eosin, 200). Immunohistochemical staining for cyclin D1 (Fig 1D) demonstrated a homogenous nuclear stain ( 100). Staging studies demonstrated stage IVB disease

Induction of Megakaryocytic Differentiation in Primary Human Erythroblasts: A Physiological Basis for Leukemic Lineage Plasticity

In myelodysplasias and acute myeloid leukemias, abnormalities in erythroid development often parallel abnormalities in megakaryocytic development. Erythroleukemic cells in particular have been shown to possess the potential to undergo megakaryocytic differentiation in response to a variety of stimuli. Whether or not such lineage plasticity occurs as a consequence of the leukemic phenotype has not previously been addressed. In this study, highly purified primary human erythroid progenitors were subjected to stimuli known to induce megakaryocytic differentiation in erythroleukemic cells. Remarkably, the primary erythroid progenitors rapidly responded with morphological and immunophenotypic evidence of megakaryocytic differentiation, equivalent to that seen in erythroleukemic cells. Even erythroblasts expressing high levels of hemoglobin manifested partial megakaryocytic differentiation. These results indicate that the lineage plasticity observed in erythroleukemic cells reflects an intrinsic property of cells in the erythroid lineage rather than an epiphenomenon of leukemic transformation.

Telomere length shortening in Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a clonal, proliferative disorder of phenotypically immature CD1a+ Langerhans cells (LC). The aetiology of LCH is unknown and data supporting an immune dysregulatory disorder as well as a clonal neoplasm have been reported. Telomere shortening has been associated with cancers and premalignant lesions as well as promoting chromosomal instability. To determine whether LCH LC have altered telomere lengths, we used dual detection of CD1a expression by immunofluorescence and telomere length by fluorescence in situ hybridization of LCH LC and lymphocytes in local, multisystem and systemic LCH and compared these with telomere lengths of LC and lymphocytes in reactive lymph nodes. LCH LC showed significantly shorter telomere lengths than LC from reactive lymph nodes or unaffected skin. Lymphocyte telomere lengths showed similar profiles among the different samples. These data show a significant telomere shortening in LCH LC in all stages of disease involvement compared with LC from reactive lymph nodes, suggesting that LCH may share mechanisms of telomere shortening and survival with clonal preneoplastic disorders and cancer, although an initiating infectious or immune event is still possible.