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// Raffaella Spina 1,3,7,* , Gessica Filocamo 2,* , Enrico Iaccino 1 , Stefania Scicchitano 1 , Michela Lupia 1 , Emanuela Chiarella 1 , Tiziana Mega 1 , Francesca Bernaudo 1 , Daniela Pelaggi 1 , Maria Mesuraca 1 , Simonetta Pazzaglia 4 , Samantha Semenkow 3 , Eli E. Bar 3,7 , Marcel Kool 5 , Stefan Pfister 5,6 , Heather M. Bond 1 , Charles G. Eberhart 3 , Christian Steinkuhler 2 , Giovanni Morrone 1 1 Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Graecia, Catanzaro, Italy. 2 Exiris srl, Via Castelfidardo 8, Rome, Italy. 3 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore MD, USA. 4 Section of Toxicology and Biomedical Sciences, ENEA CR-Casaccia, Via Anguillarese 301, S.Maria di Galeria (Roma), Italy. 5 Division of Pediatric Neurooncology, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg, Germany. 6 Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Heidelberg, Germany 7 Current address: Department of Neurological Surgery, Case Western Reserve University, Cleveland, Ohio, USA. * These authors contributed equally to this work Correspondence: Giovanni Morrone, email: // Christian Steinkuhler, email: // Charles G. Eberhart, email: // Keywords : ZNF521; gene expression; medulloblastoma; cell growth; tumorigenicity; cancer stem cells. Received : July 14, 2013 Accepted : July 25, 2013 Published : July 27, 2013 Abstract The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1 -/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1 -/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells.

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery. Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

In the past two decades it has become increasingly recognized that, in several types of cancer, only a fraction of the neoplastic cells is capable of propagating tumors upon transplantation whereas the majority of tumor cells lacks this capacity. This is particularly true for myeloid leukemias, where a minute subset of the leukemic cell population – termed leukemia stem cells (LSCs) or leukemia-initiating cells (L-ICs) - is believed to drive leukemia development and, when these cells escape chemotherapy, to cause the occurrence of relapses. The therapeutic challenges posed by this model of leukemogenesis have spurred the active quest for novel strategies for the eradication of the LSC compartment. Diverse approaches have been proposed, aimed at targeting LSCs through their surface-membrane molecules, interfering with their cell-cycle regulation, signalling pathways, DNA damage response, metabolic properties, genetic or epigenetic features, interactions with the microenvironment [1]. Such strategies have indeed led to the discovery of several candidate therapeutic agents [1-3], some of which are currently being tested in clinical trials. Systematic high-throughput screenings of collections of small molecules with therapeutic potential hold promise to yield novel effective drugs to target L-ICs, but have rarely been pursued thus far. One main limitation for this approach is the difficulty of obtaining, and propagating in culture, adequate amounts of L-ICs. This obstacle may be circumvented using experimental models of myeloid leukemogenesis based on the retro- or lentiviral transduction of normal hematopoietic stem and progenitor cells (HSPCs) with leukemia-associated oncogenes. The enforced expression of these oncogenes, alone or in combination, confers on the transduced cells features similar to those of L-ICs (including extended self-renewal and limited differentiation potential), thereby generating transformed cell lines enriched in leukemia stem-like cells. A recently-published report [4] illustrates an extensive screening carried out in the framework of a multi-institutional collaboration among the laboratories of Malcolm Moore, David Scadden, Stuart Schreiber, Benjamin Ebert and Todd Golub. This team devised a sophisticated strategy to assess the effects of almost 15,000 synthetic small molecules on the most primitive leukemic cells within the context of the bone marrow microenvironment. Murine myeloid progenitors expressing the fluorescent protein dsRed, transduced with the MLL-AF9 oncogene, were serially transplanted in irradiated hosts where they generated leukemias with increasingly short latency; LSCs were isolated from the bone marrow of quaternary recipients and co-cultured with stromal cells expressing GFP. To identify compounds selectively inhibiting LSCs but not normal HSPCs, the authors used as a readout the formation of cobblestone areas (CAs). These are clusters of small, round and phase-dark hematopoietic cells embedded in the stromal layer, derived from immature progenitors (cobblestone area-forming cells, CAFCs) that migrate and settle beneath the stroma and - after a variable latency that depends on their immaturity - begin to proliferate and generate structures that resemble cobblestones (Fig. ​(Fig.1).1). In addition to normal HSPCs, also leukemic or oncogene-transformed early progenitors can form cobblestone areas [5-6], and this property was exploited by Hartwell et al. to identify compounds with inhibitory activity on leukemic, but not normal CAFCs. As scoring CAs is extremely laborious, an automated image analysis system, trained for CA recognition, was developed to enumerate the dsRed-positive CAs in the GFP-expressing stromal monolayers. Through multiple screenings, 155 compounds were found to effectively inhibit leukemic, but not normal CAFCs, several of which with an EC50 in the low sub-micromolar range, thereby providing a pool of potentially effective anti-L-IC agents for future studies. Some of these were already known to target LSCs, like the sesquiterpene lactone, parthenolide [1]; some compounds acted exclusively on CAFCs, others exerted their inhibitory effects both through cell-intrinsic and extrinsic (stroma-mediated) mechanisms. One of the most potent and selective compounds identified was lovastatin, that was further assayed on six primary LSC-enriched human AML samples harboring different genetic aberrations. Lovastatin inhibited CAFC formation in all these samples, with an EC50 (