Sandra Durante

IKAROS deletions dictate a unique gene expression signature in patients with adult B-cell acute lymphoblastic leukemia.

Background Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult B-ALL patients (106 BCR-ABL1-positive and 38 B-ALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling. Principal Findings Total or partial deletions of IKZF1 were more frequent in BCR-ABL1-positive than in BCR-ABL1-negative B-ALL cases (75% vs 58%, respectively, p = 0.04). Comparison of the gene expression signatures of patients carrying IKZF1 deletion vs those without showed a unique signature featured by down-regulation of B-cell lineage and DNA repair genes and up-regulation of genes involved in cell cycle, JAK-STAT signalling and stem cell self-renewal. Through chromatin immunoprecipitation and luciferase reporter assays we corroborated these findings both in vivo and in vitro, showing that Ikaros deleted isoforms lacked the ability to directly regulate a large group of the genes in the signature, such as IGLL1, BLK, EBF1, MSH2, BUB3, ETV6, YES1, CDKN1A (p21), CDKN2C (p18) and MCL1. Conclusions Here we identified and validated for the first time molecular pathways specifically controlled by IKZF1, shedding light into IKZF1 role in B-ALL pathogenesis.

c-MYC oncoprotein dictates transcriptional profiles of ATP-binding cassette transporter genes in Chronic Myelogenous Leukemia CD34+ hematopoietic progenitor cells

Resistance to chemotherapeutic agents remains one of the major impediments to a successful treatment of chronic myeloid leukemia (CML). Misregulation of the activity of a specific group of ATP-binding cassette transporters (ABC) is responsible for reducing the intracellular concentration of drugs in leukemic cells. Moreover, a consistent body of evidence also suggests that ABC transporters play a role in cancer progression beyond the efflux of cytotoxic drugs. Despite a large number of studies that investigated the function of the ABC transporters, little is known about the transcriptional regulation of the ABC genes. Here, we present data showing that the oncoprotein c-MYC is a direct transcriptional regulator of a large set of ABC transporters in CML. Furthermore, molecular analysis carried out in CD34+ hematopoietic cell precursors of 21 CML patients reveals that the overexpression of ABC transporters driven by c-MYC is a peculiar characteristic of the CD34+ population in CML and was not found either in the population of mononuclear cells from which they had been purified nor in CD34+ cells isolated from healthy donors. Finally, we describe how the methylation state of CpG islands may regulate the access of c-MYC to ABCG2 gene promoter, a well-studied gene associated with multidrug resistance in CML, hence, affecting its expression. Taken together, our findings support a model in which c-MYC–driven transcriptional events, combined with epigenetic mechanisms, direct and regulate the expression of ABC genes with possible implications in tumor malignancy and drug efflux in CML. Mol Cancer Res; 9(8); 1054–66. ©2011 AACR.

Novel PLA microspheres with hydrophilic and bioadhesive surfaces for the controlled delivery of fenretinide

Abstract Novel polylactide (PLA) microspheres endowed with hydrophilic and bioadhesive surfaces as injectable formulations for the controlled release of fenretinide were prepared, using a novel technique based on the co-precipitation of PLA with gelatin, at the interface of a liquid dispersion formed by the addition of N-methylpyrrolidone containing PLA and dextrin (DX), towards an aqueous solution of gelatin (G). The resulting PLA–G–DX microspheres were compared with others prepared by the same technique using polylactide-co-glycolide (PLGA), with or without DX, and with or without phosphatidylcholine. Of the different systems, the PLA–G–DX microspheres had the best morphological, dimensional and functional characteristics. They had the highest drug loading, and their drug release was the most efficient over time without any burst effect. Their in vitro anti-tumoural activity was strongly enhanced with respect to the pure fenretinide. This paralleled the increased drug concentration inside the cells due to their marked bioadhesion to the tumour cell membranes as indicated by scanning electron microscope images.

Gelatin crosslinked with dehydroascorbic acid as a novel scaffold for tissue regeneration with simultaneous antitumor activity

A porous scaffold was developed to support normal tissue regeneration in the presence of residual tumor disease. It was prepared by gelatin crosslinked with dehydroascorbic acid (DHA). A physicochemical characterization of the scaffold was carried out. SEM and mercury porosimetry revealed a high porosity and interconnection of pores in the scaffold. Enzymatic degradation provided 56% weight loss in ten days. The scaffold was also evaluated in vitro for its ability to support the growth of normal cells while hindering tumor cell development. For this purpose, primary human fibroblasts and osteosarcoma tumor cells (MG-63) were seeded on the scaffold. Fibroblasts attached the scaffold and proliferated, while the tumor cells, after an initial attachment and growth, failed to proliferate and progressively underwent cell death. This was attributed to the progressive release of DHA during the scaffold degradation and its cytotoxic activity towards tumor cells.

Abstract 906: Gas1 and Kif27 genes are strongly up-regulated biomarkers of Hedgehog inhibition (PF-04449913) on leukemia stem cells in Phase I Acute Myeloid Leukemia and Chronic Myeloid Leukemia treated patients

Hedgehog (Hh) pathway activation contributes to leukemia development and growth, and that targeted pathway inhibition is likely to offer an efficient therapeutic opportunity. PF-04449913, a Hh pathway inhibitor, is a new selective and potent inhibitor of leukemia self-renewal and is currently being evaluated in phase I clinical trials. In order to identify new potential clinical biomarkers for the PF-04449913, we studied CD34+ leukemia stem cell population (LSC) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies. This experimental clinical trial enrolled Myelofibrosis (MF), MDS, blastic phases CML, chronic myelomonocytic leukemia (CMML) and AML patients (pts). We were able to collect and separate highly purified (98%) bone marrow CD34+ cells from 5 AML, 1 MF and 2 CML pts by immunomagnetic separation, and analysed them for gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. Clustering of their expression profiles showed that mostly genes differentially expressed are mainly related to Hh signaling, this providing further evidences that PF-04449913 really therapeutically targets the Hh pathway. Regarding genes involved in Hh signaling pathway, Gas1 and Kif27 were strongly upregulated (fold change 1.0947 and 1.12757 respectively; p-value 0.01 and 0.02 respectively) in CD34+ LSC after 28 days exposure to PF-04449913 as compared to baseline, suggesting these two genes have potential as new biomarkers of activity. GAS-1 is a Sonic Hedgehog (Shh)-binding protein; it acts to sequester Shh and inhibit the Shh signalling pathway. Kif27 mainly acts as a negative regulator in the Hh signaling pathway, and inhibits the transcriptional activator activity of Gli1 by inhibiting its nuclear translocation. Other genes were differentially expressed after ‘ex- vivo’ treatment with PF-04449913 as compared to baseline: we observed a down regulation of Bcl2 (fold change -1.03004), ABCA2 (fold change -1.08966), LEF1 (fold change -1.28457), Gli1 (fold change -1.0775), Smo (fold change -1.07702), and an upregulation of Gli2 (fold change 1.08191). Conclusions: This data demonstrates that PF-04449913 specifically targets the Hh Pathway in CD34+ cells, suggesting that Hh inhibition may impair leukemia stem cell maintenance. In addition, we identify several new potential biomarkers (e.g. Gas1 and KIF27). Taken together, these data may be useful for pts selection strategies and subsequent eradication of the LSC. Acknowledgments: Work supported by Pfizer, European LeukemiaNet, FIRB 2006, AIRC, AIL, COFIN, University of Bologna and BolognAIL. 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 906. doi:1538-7445.AM2012-906

Influence of a biomimetic gelatin porous scaffold in chondrogenic and osteogenic differentiation of mesenchymal stem cells

Recently, tissue engineering has merged with stem cell technology to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries/disorders because of the low self-regenerating capacity of the matrix secreting cells, particularly chondrocytes (1). Gelatin based scaffolds are considered to be a highly suitable 3D material for tissue regeneration, due to high biocompatibility and adaptation to native tissues. In the present study, the chondrogenic and osteogenic potential of a porous gelatin based scaffold (2), alone or in combination with hydroxyapatite crystals, was investigated in human mesenchymal stem cells. Cells were culture up to 4 weeks on the scaffold and on monolayer. MTT assay was performed to evaluate cell viability, light and transmission electron microscopy were carried out to demonstrate cell colonization inside the porous architecture of the biomaterial and scaffold adhesion. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan and versican and osteogenic markers such as Collagen type I, Runx -2, osteopontin and bone matrix protein, were investigated by Real Time PCR. Results showed an high cell viability, adhesion and colonization of the scaffold. Real Time PCR data demonstrated an up-regulation of all the chondrogenic and osteogenic markers analyzed. In conclusion, gelatin porous scaffold provides an improved environment for chondrogenic and osteogenic differentiation of stem cells compared to cell monolayer culture system.

Ultrastructural changes in human gingival fibroblasts after exposure to 2-hydroxy-ethyl methacrylate.

Polymerized resin-based materials are successfully utilized in medical applications. One draw- back is the release of monomers from the matrix due to an incomplete polymerization or degradation processes. Released monomers can diffuse in the systemic circulation and induceadverse effects to biological tissues. Although there are many hypotheses about the induction of cell death by resin monomers, the underlying mechanisms are still under discussion. The aim of the study was to investigate the morphological modifications in human gingival fibroblasts exposed to 2-hydroxy-ethyl methacrylate (HEMA) to better elucidate the mechanism of cell death induced by resin monomers. Primary cultures of gingival fibroblasts were exposed to 3mM HEMA for 24 h, 72 h, 96 h. Morphological investigations were performed by scanning and transmission electron microscopy, while western blot for caspase-3 was carried out to ver- ify apoptosis. Electron microscopy images showed deep changes in the cell surface and cyto- plasm after 72 h and 96 h of HEMA treatment. Autophagic vesicles were easily observed just after 24 h. Cleaved caspase-3 was detected after 72 h of treatment. These findings suggest that resin based materials induced cell death by the cooperation of apoptosis and autophagy mecha- nisms. The understanding of these mechanisms will lead to the development of smart biomate- rials without or with low adverse effects.

Changes of microenvironment in an in vitro co-culture system

For the enhanced understanding of fundamental cancer biology and improving molecular diagnostics and therapeutics, the role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance (1, 2). The aim of this study was to establish an in vitro model based on a co-culture of healthy human fibroblasts (HFs) and human osteosarcoma cells (MG-63s) to simulate the microenvironment including tumor and healthy cells. The HFs and MG-63s were in vitro co-cultured for a period of time ranging from 24h to 7 days. Cell morphology and organization were studied by phase contrast microscopy while the expression of Human Cartilage Glycoprotein 39 (YKL-40), Vascular Endothelial Growth Factor (VEGF), Matrix Metalloprotease 1 (MMP1) and IL1 alpha was investigated by Real Time PCR and Western Blotting. To better correlate the role of YKL-40, VEGF, MMP1 and IL1 alpha, siRNA knockdown of YKL-40 in MG-63 cells was performed, and changes in protein expression in the co-cultures were analyzed. Results showed a characteristic disposition of tumor and healthy co-cultured cells in columns which are not visible in tumor and healthy cells grown singularly. The expression of YKL-40, VEGF and MMP1 significantly changed in co-cultured cells compared to HFs and MG-63s separately cultured. siRNA knockdown of YKL-40 was responsible of a down-regulation of VEGF expression on HFs, and an increase of IL1 alpha expression in MG-63s. These results suggest that the tumor microenvironment has an influence on the protein expression of the healthy surrounding tissues and the process of tumorigenicity.

Matricellular protein expression as a new parameter to test in vitro cytotoxicity of a 3D biomimetic material

Up regulation of matricellular proteins are often involved in in-vitro cell suffering and in in-vivo encapsulation, foreign body reaction and inflammation response following scaffold implantation [1]. The cytotoxicity of biomaterials is generally tested according to the ISO standard 10993-5, based mainly on viability tests. Further assays, based on the improved biocompatibility knowledge, could be suggested to better analyze the cytotoxicity of implant materials. The aim of the study was to propose matricellular protein expression as a new assay in the evaluation of cytotoxicity of implant materials. Tenascin C, osteocalcin and osteopontin belonged to the matricellular protein family and they were chosen as cytotoxicity markers. Viability test, Real Time PCR and western blot in mesenchymal stem cells seeded on collagen/hydroxyapatite scaffold were performed to evaluate gene/protein expression. Transmission electron microscopy was carried out to evaluate morphological changes induced by cell/scaffold interactions. Results showed an high viability of the cells during the time of culture and a good cell adhesion and morphology observed by electron microscopy. A lower expression of tenascin-c, osteonectin and osteopontin compared to cells cultured on tissue flasks was demonstrated both by Real Time PCR and western blot suggesting a better cell culture condition in 3D biomimetic scaffold compared to tissue flasks. Based on our results, we propose the analysis of matricellular protein expression as a new parameter for testing cytotoxicity of implant materials.

Cell adhesion and proliferation on gelatin-ascorbic acid scaffold

The aim of this study was to combine gelatin and ascorbic acid to produce a scaffold with antitumoral properties. Gelatin was chosen because is a derivative of collagen, the major constituent of skin, bones and connective tissues, and because it does not exhibit antigenicity. Ascorbic acid had been reported for many years to have varying effects on tumor cells, using models such as hepatoma, pancreatic and colon cancer, sarcoma, leukemia, prostate cancer and mesothelioma. The chemical, physical and biological properties of this gelatin/ascorbic acid scaffold, including morphology by high resolution SEM analysis, swelling ratio and degradation rate were studied. Cell proliferation assay was performed to compare osteosarcoma cell line (MG-63) and human fibroblast cells (HGF), in order to verify the effects of the scaffold on cell growth, proliferation and cell adhesion. The scaffold was hydrated in cell medium for 24 h. The swelling ratio and degradation rate were performed starting from 1 to 7 days. MG-63 cells and HGFs were seeded on hydrated scaffolds and MTT test and high resolution SEM analysis were performed at 24hr, 3, 7 and 10 days. The swelling ratio showed an absorption of about 10 times of its dry weight and a degradation starting from 6 days. High resolution SEM analysis revealed a good porosity, that facilitated the attachment of cells and their migration inside the scaffold. MTT test showed a different proliferation pattern for the two cellular types. The results proved that gelatin/ascorbic acid scaffold has properties of biocompatibility and has a different effect on cell proliferation.