Paula Scotland

The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells

In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer.

The PICALM Protein Plays a Key Role in Iron Homeostasis and Cell Proliferation

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimers disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM’s function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.

A CALM-derived nuclear export signal is essential for CALM-AF10–mediated leukemogenesis

The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10-driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10-dependent transformation. Fusions of the CALM NES (NES(CALM)-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10-dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.

A cord blood monocyte–derived cell therapy product accelerates brain remyelination

Microglia and monocytes play important roles in regulating brain remyelination. We developed DUOC-01, a cell therapy product intended for treatment of demyelinating diseases, from banked human umbilical cord blood (CB) mononuclear cells. Immunodepletion and selection studies demonstrated that DUOC-01 cells are derived from CB CD14+ monocytes. We compared the ability of freshly isolated CB CD14+ monocytes and DUOC-01 cells to accelerate remyelination of the brains of NOD/SCID/IL2Rγnull mice following cuprizone feeding-mediated demyelination. The corpus callosum of mice intracranially injected with DUOC-01 showed enhanced myelination, a higher proportion of fully myelinated axons, decreased gliosis and cellular infiltration, and more proliferating oligodendrocyte lineage cells than those of mice receiving excipient. Uncultured CB CD14+ monocytes also accelerated remyelination, but to a significantly lesser extent than DUOC-01 cells. Microarray analysis, quantitative PCR studies, Western blotting, and flow cytometry demonstrated that expression of factors that promote remyelination including PDGF-AA, stem cell factor, IGF1, MMP9, MMP12, and triggering receptor expressed on myeloid cells 2 were upregulated in DUOC-01 compared to CB CD14+ monocytes. Collectively, our results show that DUOC-01 accelerates brain remyelination by multiple mechanisms and could be beneficial in treating demyelinating conditions.

Expanding and Streamlining Cord Tissue‐Derived Mesenchymal Stem Cells Manufacture

10 Cord tissue-derived mesenchymal stem cells (CT-MSC) are increasingly being used in clinical studies to address a variety of health issues and diseases due to their tissue-regenerating and immune-modulatory properties. With this promise of therapies comes the need for the manufacturing of a large number of CTMSCs for dosing. At the Marcus Center for Cellular Cures at Duke, we have created a process for the manufacturing of large batches of CT-MSCs. The path from first isolating CT-MSC progenitors from cord tissue to delivery of the appropriate CT-MSC dose at the treatment site comprises many steps that need to be efficiently connected while conforming to current Good Manufacturing Practice standards. The process starts with the isolation of CT-MSC progenitors from donated cord tissue using enzymatic digestion in a tissue dissociator and subsequent culturing in flasks. We have optimized the timing of supplement addition to CT-MSC growth media and cell harvest for obtaining populations of pure CT-MSC. Expansion of those MSCs were initially carried out in cell culture flasks and then broadened to multilayer stacked flask systems in two expansion steps. In order to increase batch size, improve automation, and reduce the number of cell passages, we currently are testing cell expansion in large closed system bioreactors. Parameters tested include loading conditions, media, feed rates, and harvest conditions. Scaling up cell production also has to be matched with up-scaled procedures for cell washing, addition of cryopreservation media, rapid dispensing into suitable cryopreservation containers, and cell cryopreservation. We will briefly review the factors and equipment used at our facility to complete the scaled-up CTMSC process. Supplying the demand of high numbers of quality CT-MSC product requires a manufacturing process that is closedsystem, automated, and efficient in time and cost throughout all steps. We are developing a manufacturing process that will comply with these requirements and that will provide cells for the increasing demand of MSC clinical trials. STEM CELLS TRANSLATIONAL MEDICINE | StemCellsTM.com

Gene products promoting remyelination are up-regulated in a cell therapy product manufactured from banked human cord blood

BACKGROUND AIMS DUOC-01, a cell product being developed to treat demyelinating conditions, is composed of macrophages that arise from CD14+ monocytes in the mononuclear cell (MNC) population of banked cord blood (CB). This article demonstrates that expression of multiple gene products that promote remyelination is rapidly up-regulated during manufacturing of DUOC-01 from either MNC or purified CB CD14+ monocytes. METHODS Cell cultures were initiated with MNC or with immunoselected CD14+ monocytes isolated from the same CB unit. Cell products present in these cultures after 2 and 3 weeks were compared by three methods. First, quantitative polymerase chain reaction was used to compare expression of 77 transcripts previously shown to be differentially expressed by freshly isolated, uncultured CB CD14+ monocytes and DUOC-01. Second, accumulation of 16 soluble proteins in the culture medium was measured by Bioplex methods. Third, whole transcriptomes of the cell products were compared by microarray analysis. RESULTS Key transcripts in multiple pathways that promote remyelination were up-regulated in DUOC-01, and substantial secretion of proteins corresponding to many of these transcripts was detected. Cell products manufactured from MNC or from CD14+ monocytes were similar with regard to all metrics. Upregulation of gene products characteristic of DUOC-01 was largely completed within 14 days of culture. CONCLUSION We demonstrate that expression of multiple gene products that promote remyelination is up-regulated during the first 2 weeks of manufacturing of DUOC-01. Measuring these mechanistically important transcripts and proteins will be useful in monitoring manufacturing, evaluating manufacturing changes, and developing mechanism-based product potency assays.

Abstract LB-154: Nuclear export of CALM-AF10 is essential for leukemogenesis

Background: The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is recurrently found in patients with aggressive and difficult-to-treat hematopoietic malignancies (AML and T-ALL). CALM-AF10 leukemias are characterized by upregulation of Hoxa cluster genes and a reduction in H3K79 methylation. The native AF10 protein localizes to the nucleus, while native CALM protein is found predominantly in the cytoplasm. It has been shown that the OM-LZ domain of AF10 interacts with the histone methyltransferase, Dot1L, and that this interaction is necessary for CALM-AF10-mediated transformation. Although it is known that CALM functions as an accessory endocytic protein, the precise role of CALM in leukemogenesis is unclear. Objective: To determine the role of CALM in CALM-AF10-mediated transformation in an effort to identify novel therapeutic targets. Results: Through structure-function analyses, we determined that a minimal region of CALM (aa 520-583) is critical for CALM-AF10-mediated transformation of murine hematopoietic progenitor cells (HPCs). Further analysis of this sequence identified a nuclear export signal (NES) encoded by CALM aa 544-553. The CALM NES mediates the cytoplasmic localization of CALM-AF10; point mutation of the CALM NES (NES*) results in exclusive CALM(NES*)-AF10 accumulation in the nucleus. In contrast to CALM-AF10, transduction of primary murine HPCs with CALM(NES*)-AF10 fails to cause in vitro transformation. Fusion of an 18 aa peptide that includes the CALM NES in frame with AF10 (NESCALM-AF10) is sufficient to induce transformation in vitro and leukemogenesis in vivo. Leukemias induced by NESCALM-AF10 recapitulated the phenotype of CALM-AF10 leukemias, including aberrant Hoxa cluster upregulation and decreased H3K79 methylation. Strikingly, fusion of the NES from different proteins, such as PKI-α and APC, to AF10 also conferred cytoplasmic localization and resulted in transformation in vitro. Finally, human CALM-AF10 leukemia cell lines (U937 and P31-FUJI) display increased sensitivity to a compound that inhibits nuclear export, Leptomycin B. Conclusions: We have determined that a CALM-derived nuclear export signal is both necessary and sufficient for CALM-AF10-mediated transformation. These findings reveal a novel mechanism by which nuclear export of CALM-AF10 mediates leukemogenesis. We postulate that nuclear export inhibitor (NEI) compounds may offer new therapeutic possibilities for patients with aggressive CALM-AF10 leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-154. doi:1538-7445.AM2012-LB-154

Abstract 3952: CALM deficiency in fit1 embryonic cells alters expression and rate of endocytosis of growth factor receptors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Gene rearrangements involving the Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) and Mixed-Lineage Leukemia (MLL) or AF10 genes have been identified in aggressive leukemias and lymphomas. Expression of CALM-containing fusion proteins immortalizes murine hematopoietic cells in vitro, correlating with leukemogenesis in vivo. Disruption of normal CALM, MLL or AF10 protein function as a result of these translocations likely contributes to transformation, although the precise mechanisms are unknown. The native CALM protein is involved in clathrin-mediated endocytosis (CME); it localizes to the cytoplasmic side of endocytic vesicles, interacts with both membrane elements and clathrin, and when over- or under-expressed, disrupts endocytosis. To better understand the effects of altered CALM activity, we have focused our studies on mutant fit1 mice that lack CALM expression. Previous studies have shown that these mice have iron deficiency, defective hematopoiesis and ultimately premature death. Here we examine CALMs role in endocytosis and cellular function using cells derived from fit1 mice. Objective: To examine the effect of CALM deficiency on cell surface receptor expression and endocytosis using mouse embryonic fibroblasts (MEFs) and fetal liver cells derived from normal and CALM-deficient mice. Design: Day 14 MEFs were generated from normal, heterozygous, and mutant fit1 embryos. Immortalized MEFs were compared in terms of quantity of cell surface and total cellular receptors (by flow cytometry and Western blotting), receptor mRNA levels, and rate of endocytosis. Receptors were also measured in fetal liver derived from the same embryos. Results: When compared to their normal counterparts, cells lacking full length CALM showed altered cell surface receptor expression: transferrin receptor (TfR) protein levels measured by flow cytometry were increased two-fold, while EGF receptor (EGFR) levels were reduced two-fold. These results correlated with increased total cellular TfR protein and mRNA levels and decreased total EGFR protein levels. Whereas the rate of internalization of TfR was similar, the rate of endocytosis of EGFR was significantly reduced in CALM-deficient compared with wildtype cells. Elevated TfR levels were also identified in CALM-deficient fetal liver cells. Conclusions: CALM deficiency in fit1-derived cells results in increased TfR surface expression; this is consistent with the iron deficiency phenotype of fit1 mice. The reduced surface expression of EGFR seen in fit1 cells may be a compensatory mechanism that counteracts the increased growth factor receptor signaling that is associated with impaired endocytosis. Since CALM haploinsufficiency is a feature of CALM-AF10 and MLL-CALM leukemias, our results suggest that the perturbation of normal growth factor biology may contribute to transformation in these malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3952.