Roberto Tonelli

Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis

We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH2 terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G1-phase accumulation (39–53%) in IMR-32 and apoptosis (56% annexin V–positive cells at 24 hours in IMR-32 and 22% annexin V–positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA–based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.

The MYCN Oncogene as a Specific and Selective Drug Target for Peripheral and Central Nervous System Tumors

MYCN belongs to the MYC family of proto-oncogenes, which encode for transcription factors of the basic-helix-loop-helix-zipper (bHLHZ) class and is fundamental in the development of the peripheral and central nervous systems (PNS and CNS). While Myc is ubiquitous, MYCN has a very restricted expression pattern: it is mainly expressed during embryonic development, but then becomes downregulated, while in adults it is usually detected in B-cell development. Identification of selective inhibitors of MYCN and its mRNA and protein could be important for the development of more specific, effective and less toxic therapeutic agents for tumors of the PNS and CNS. In children, the most common tumors of the PNS and CNS are neuroblastomas and medulloblastomas, respectively. About 30% of neuroblastoma (NB) tumors present MYCN amplification/over-expression, which is associated with rapid progression and poor prognosis. N-Myc is essential during neurogenesis for the rapid expansion of progenitor cells in the brain. MYCN amplification and over-expression has been reported in medulloblastoma, and especially in the desmoplastic type. Other tumors associated with MYCN overexpression include retinoblastoma, small cell lung carcinoma, glioblastoma and certain embryonal tumors. A cell-based, N-Myc-dependent luciferase reporter gene assay to identify specific N-Myc small-molecule inhibitors has allowed identification of five compounds showing significant activity. Antisense oligodeoxynucleotides have been shown to inhibit N-Myc production and anti-tumoral activity in vitro and in vivo for NB. Peptide nucleic acids (PNA), which belong to the most recent (third) generation of nucleic acid therapeutics, form highly stable duplexes with DNA and RNA, and are resistant to degradation by nucleases and proteases. Encouraging results have been reported utilizing a PNA-based antisense strategy for inhibition of N-Myc expression in neuroblastoma.

Antitumor Activity of Sustained N-Myc Reduction in Rhabdomyosarcomas and Transcriptional Block by Antigene Therapy

Purpose: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription. Experimental Design: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined. Results: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity. Conclusion: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo. Clin Cancer Res; 18(3); 796–807. ©2011 AACR.

Mutational screening of thrombopoietin receptor gene (c-mpl) in patients with congenital thrombocytopenia and absent radii (TAR)

Thrombocytopenia with absent radii (TAR) is a rare autosomal recessive disease characterized by hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. We performed mutational screening of coding and promoter regions of the c‐mpl gene, encoding thrombopoietin (TPO) receptor, by sequence analysis in four unrelated patients affected by TAR syndrome. Our results indicate that c‐mpl gene mutations are not a common cause of thrombocytopenia in TAR syndrome.

MLL-AF9 oncogene expression affects cell growth but not terminal differentiation and is downregulated during monocyte-macrophage maturation in AML-M5 THP-1 cells.

The MLL-AF9 oncogene – one of the most frequent MLL/HRX/ALL-1 rearrangements found in infantile and therapy-related leukaemias – originates from t(9;11)(p22;q23) and is mainly associated with monocytic acute myeloid leukaemia (AML-M5; FAB-classification). Here, we investigated the MLL-AF9 function by means of an antisense phosphorothioate-oligodeoxyribonucleotide (MLL-AF9-PS-ODNas) using the THP-1 AML-M5 cell line carrying t(9;11). Having confirmed that MLL-AF9-PS-ODNas induces strong inhibition of THP-1 cell growth, but only a moderate increase in apoptosis, we found that MLL-AF9-PS-ODNas did not induce morpho-functional terminal differentiation or restore M-CSF-, G-CSF- or GM-CSF-induced differentiation. Moreover, THP-1 cells showed the same phenotype with/without MLL-AF9-PS-ODNas. In THP-1 cells differentiated to mature macrophage-like cells by PMA/TPA or ATRA, MLL-AF9 expression was downregulated. Thus, in the monocytic lineage, MLL-AF9 may be expressed only in early phases and can induce deregulated amplification in both nonmalignant and malignant cells, maintaining the monocytic phenotype without blocking final maturation. Our findings suggest that: (1) as well as directly promoting cell growth, MLL-AF9 may also indirectly determine phenotype; (2) other leukaemogenic mutations associated with MLL-AF9-related leukaemias should be searched for mainly in processes of resistance to apoptosis (where MLL-AF9 may play only a limited role) and differentiation blockage (where MLL-AF9 may play no role).

Long-term bone marrow cultures in Diamond-Blackfan anemia reveal a defect of both granulomacrophage and erythroid progenitors.

The hematopoietic defect of Diamond-Blackfan anemia (DBA) results in selective failure of erythropoiesis. Thus far, it is not known whether this defect originates from an intrinsic impediment of hematopoietic progenitors to move forward along the erythroid pathway or to the impaired capacity of the bone marrow (BM) microenvironment to support proliferation and differentiation of hematopoietic cells. Reduced longevity of long-term bone marrow cultures, the most physiologic in vitro system to study the interactions of hematopoietic progenitors and hematopoietic microenvironment, is consistent with a defect of an early hematopoietic progenitor in DBA. However, stromal adherent layers from DBA patients generated in a long-term culture system, the in vitro counterpart of BM microenvironment, did not show evidence of any morphologic, phenotypic, or functional abnormality. Our major finding was an impaired capacity of enriched CD34+ BM cell fraction from DBA patients, cultured in the presence of normal BM stromal cells, to proliferate and differentiate along the erythroid pathway. A similar impairment was observed in some DBA patients along the granulomacrophage pathway. Our result points to an intrinsic defect of a hematopoietic progenitor with bilineage potential that is earlier than previously suspected as a relevant pathogenetic mechanism of the disease. The finding of impaired granulopoiesis in some DBA patients underlines the heterogeneity of this rare disorder.

Generation and ex vivo expansion of cytotoxic T lymphocytes directed toward different types of leukemia or myelodysplastic cells using both HLA-matched and partially matched donors

OBJECTIVE Successful priming and in vitro expansion of anti-leukemia cytotoxic T lymphocytes (CTL) are preliminary conditions for designing approaches of adoptive immunotherapy in patients with hematological malignancies undergoing allogeneic hematopoietic stem cell transplantation (HSCT). In this study, we evaluated the possibility of generating and expanding in vitro CTL directed toward different types of either leukemia or myelodysplastic cells, using both HLA-matched and partially matched donors. PATIENTS AND METHODS Eleven donor/recipient pairs were enrolled; donor-derived dendritic cells, pulsed with patient blast cells, were used to generate CTL. RESULTS Anti-leukemia CTL lines were successfully obtained from 10 of 11 donors. After repeated rounds of stimulation, CTL lines showed, along with an increase in cytotoxic activity, a variable but continuous expansion of cultured cells. In order to increase the magnitude of CTL expansion, two anti-leukemia CTL lines were further stimulated using allogeneic feeder cells, anti-CD3, and low doses of interleukin-2 (IL-2). This stimulation gave rise to 150-fold to 270-fold expansion of the absolute number of cultured cells. Most cultures showed either absent or low reactivity of anti-leukemia CTL against patient non-leukemia cells. Three anti-leukemia CTL lines displayed a more pronounced cytotoxicity against nonmalignant recipient cells, which was always lower than that observed against leukemia blasts (LB). Spectratyping analysis of the TCR-Vbeta subfamilies revealed a preferential expansion of oligoclonal populations that persisted in CTL lines following repeated rounds of stimulation. CONCLUSIONS Results provide the biological background for designing protocols of adoptive immunotherapy for the control of minimal residual disease in patients with hematological malignancies given HSCT.

In Vivo Bioluminescence Imaging of Murine Xenograft Cancer Models with a Red-shifted Thermostable Luciferase

PurposeConventional in vivo bioluminescence imaging using wild-type green-emitting luciferase is limited by absorption and scattering of the bioluminescent signal through tissues. Imaging methods using a red-shifted thermostable luciferase from Photinus pyralis were optimized to improve the sensitivity and image resolution. In vivo bioluminescence imaging performance of red- and green-emitting luciferases were compared in two different xenograft mouse models for cancer.MethodsHuman hepatoblastoma cell line (HepG2) and human acute monocytic leukemia cell line (Thp1) cells were genetically engineered using retroviral vector technology to stably express the red-shifted or the wild-type green luciferase. A xenograft model of liver cancer was established by subcutaneous injection of the HepG2-engineered cells in the flank regions of mice, and a leukemia model was generated by intravenous injection of the engineered Thp1 cells. The cancer progression was monitored with an ultrasensitive charge-coupled device camera. The relative intensities of the green- and red-emitting luciferases were measured, and the resulting spatial resolutions of the images were compared. Imaging was performed with both intact and scarified live animals to quantify the absorption effects of the skin and deep tissue.ResultsThe red-emitting luciferase was found to emit a bioluminescence signal with improved transmission properties compared to the green-emitting luciferase. By imaging the HepG2 models, which contained tumors just beneath the skin, before and after scarification, the percentage of light absorbed by the skin was calculated. The green bioluminescent signal was 75 ± 8% absorbed by the skin, whereas the red signal was only 20 ± 6% absorbed. The Thp1 model, which contains cancer cells within the bones, was likewise imaged before and after scarification to calculate the percentage of light absorbed by all tissue under the skin. This tissue was responsible for 90 ± 5% absorption of the green signal, but only 65 ± 6% absorption of the red signal.ConclusionTwo different bioluminescent mouse cancer models demonstrate the utility of a new red-shifted thermostable luciferase, Ppy RE-TS, that improved the in vivo imaging performance when compared with wild-type P. Pyralis luciferase. While wild-type luciferase is currently a popular reporter for in vivo imaging methods, this study demonstrates the potential of red-emitting firefly luciferase mutants to enhance the performance of bioluminescence imaging experiments.

An erythroid and megakaryocytic common precursor cell line (B1647) expressing both c‐mpl and erythropoietin receptor (Epo‐R) proliferates and modifies globin chain synthesis in response to megakaryocyte growth and development factor (MGDF) but not to erythropoietin (Epo)

A human megakaryocyte cell line (B1647) has been established from bone marrow cells obtained from a patient with acute myelogenous leukaemia (FAB M2). The cells were CD34−, CD33+, HLA‐DR+, CD38+, and expressed the immunophenotypic markers of the megakaryocyte lineage (CD41 and von Willebrand factor). Moreover the cells expressed the c‐mpl (thrombopoietin receptor) mRNA and protein. On the other hand, the B1647 cells also possessed erythroid lineage characteristics: the vast majority of cells were glycophorin positive, and about 10% of unstimulated cells stained with an anti‐globin γ chain MoAb. In addition, S1 protection analysis demonstrated expression of β‐globin mRNA, and Epo receptor (Epo‐R) protein was detected by cytofluorimetric assay. Several growth factors, when tested alone or in combination, failed to influence the B1647 cell growth. A significant increase of cell proliferation was observed only after the addition, in serum‐free culture, of recombinant human megakaryocyte growth development factor (MGDF), a recombinant c‐mpl ligand encompassing the receptor‐binding domain and identical to thrombopoietin (TPO), at concentrations ranging from 0.01 to 1 ng/ml. Interestingly, MGDF failed to induce megakaryocytic differentiation of the B1647 cells, but significantly increased the synthesis of the globin γ‐chain.

Ex vivo generation and expansion of anti-tumor cytotoxic T-cell lines derived from patients or their HLA-identical sibling

Successful ex‐vivo priming and long‐term maintenance of anti‐tumor cytotoxic T‐cell (CTL) lines are preliminary conditions for their use in approaches of adoptive immunotherapy for patients with cancer. We describe the results of a novel procedure for generating in vitro anti‐tumor CTL using CD8‐enriched peripheral blood mononuclear cells (PBMC) and dendritic cells (DC), pulsed with irradiated tumor cells (TC) as source of tumor antigen. Eight patients were enrolled in our study: 4 sarcoma, 2 renal cell carcinoma, 1 ovarian carcinoma and 1 breast carcinoma. Ten anti‐tumor CTL‐lines cytotoxic towards patient TC were generated. Five CTL‐lines were obtained using both DC and PBMC from the patients (autologous setting). For 5 CTL‐lines, DC derived from an HLA‐identical sibling were employed (allogeneic setting): patients or siblings PBMC were used to generate CTL‐lines in 2 and 3 cases, respectively,. After tumor‐specific rounds of stimulation, followed by antigen‐independent cycle of expansion, CTL‐lines obtained in both autologous and allogeneic setting showed an expansion of the absolute number of cultured cells. In 6 of 10 CTL‐lines, the majority of effector cells (>70%) were CD3+/CD8+, while in the remaining 4, 40–70% of effector cells were CD3+/CD4+. Both CD8+ and CD4+ T cells displayed anti‐tumor cytotoxic activity. Spectratyping analysis of the TCR‐Vβ subfamilies revealed a preferential expansion of oligoclonal populations in 18 of 24Vβ subfamily. Altogether these results demonstrate that our experimental approach is suitable for efficiently generating and expanding anti‐solid tumor CTL to be used for adoptive immunotherapy.