Maria Artesi

Early termination of ISRCTN45828668, a phase 1/2 prospective, randomized study of Sulfasalazine for the treatment of progressing malignant gliomas in adults

BackgroundSulfasalazine, a NF-kappaB and x(c)-cystine/glutamate antiport inhibitor, has demonstrated a strong antitumoral potential in preclinical models of malignant gliomas. As it presents an excellent safety profile, we initiated a phase 1/2 clinical study of this anti-inflammatory drug for the treatment of recurrent WHO grade 3 and 4 astrocytic gliomas in adults.Methods10 patients with advanced recurrent anaplastic astrocytoma (n = 2) or glioblastoma (n = 8) aged 32-62 years were recruited prior to the planned interim analysis of the study. Subjects were randomly assigned to daily doses of 1.5, 3, 4.5, or 6 grams of oral sulfasalazine, and treated until clinical or radiological evidence of disease progression or the development of serious or unbearable side effects. Primary endpoints were the evaluation of toxicities according to the CTCAE v.3.0, and the observation of radiological tumor responses based on MacDonald criteria.ResultsNo clinical response was observed. One tumor remained stable for 2 months with sulfasalazine treatment, at the lowest daily dose of the drug. The median progression-free survival was 32 days. Side effects were common, as all patients developed grade 1-3 adverse events (mean: 7.2/patient), four patients developed grade 4 toxicity. Two patients died while on treatment or shortly after its discontinuation.ConclusionAlthough the proper influence of sulfasalazine treatment on patient outcome was difficult to ascertain in these debilitated patients with a large tumor burden (median KPS = 50), ISRCTN45828668 was terminated after its interim analysis. This study urges to exert cautiousness in future trials of Sulfasalazine for the treatment of malignant gliomas.Trial RegistrationCurrent Controlled Trials ISRCTN45828668

Connexin 30 expression inhibits growth of human malignant gliomas but protects them against radiation therapy

BACKGROUND Glioblastomas remain ominous tumors that almost invariably escape treatment. Connexins are a family of transmembrane, gap junction-forming proteins, some members of which were reported to act as tumor suppressors and to modulate cellular metabolism in response to cytotoxic stress. METHODS We analyzed the copy number and expression of the connexin (Cx)30 gene gap junction beta-6 (GJB6), as well as of its protein immunoreactivity in several public and proprietary repositories of glioblastomas, and their influence on patient survival. We evaluated the effect of the expression of this gap junction protein on the growth, DNA repair and energy metabolism, and treatment resistance of these tumors. RESULTS The GJB6 gene was deleted in 25.8% of 751 analyzed tumors and mutated in 15.8% of 158 tumors. Cx30 immunoreactivity was absent in 28.9% of 145 tumors. Restoration of Cx30 expression in human glioblastoma cells reduced their growth in vitro and as xenografts in the striatum of immunodeficient mice. Cx30 immunoreactivity was, however, found to adversely affect survival in 2 independent retrospective cohorts of glioblastoma patients. Cx30 was found in clonogenic assays to protect glioblastoma cells against radiation-induced mortality and to decrease radiation-induced DNA damage. This radioprotection correlated with a heat shock protein 90-dependent mitochondrial translocation of Cx30 following radiation and an improved ATP production following this genotoxic stress. CONCLUSION These results underline the complex relationship between potential tumor suppressors and treatment resistance in glioblastomas and single out GJB6/Cx30 as a potential biomarker and target for therapeutic intervention in these tumors.

Cis-perturbation of cancer drivers by the HTLV-1/BLV proviruses is an early determinant of leukemogenesis

Human T-cell leukaemia virus type-1 (HTLV-1) and bovine leukaemia virus (BLV) infect T- and B-lymphocytes, respectively, provoking a polyclonal expansion that will evolve into an aggressive monoclonal leukaemia in ∼5% of individuals following a protracted latency period. It is generally assumed that early oncogenic changes are largely dependent on virus-encoded products, especially TAX and HBZ, while progression to acute leukaemia/lymphoma involves somatic mutations, yet that both are independent of proviral integration site that has been found to be very variable between tumours. Here, we show that HTLV-1/BLV proviruses are integrated near cancer drivers which they affect either by provirus-dependent transcription termination or as a result of viral antisense RNA-dependent cis-perturbation. The same pattern is observed at polyclonal non-malignant stages, indicating that provirus-dependent host gene perturbation contributes to the initial selection of the multiple clones characterizing the asymptomatic stage, requiring additional alterations in the clone that will evolve into full-blown leukaemia/lymphoma.

Casein kinase 2 inhibition modulates the DNA damage response but fails to radiosensitize malignant glioma cells

Inhibitors of casein kinase 2 (CK2), a regulator of cell proliferation and mediator of the DNA damage response, are being evaluated in clinical trials for the treatment of cancers. Apigenin was capable of inhibiting the activation of CK2 following γ irradiation in LN18 and U87 malignant glioma cells. Apigenin and siRNA-mediated CK2 protein depletion further inhibited NF-κB activation and altered the Tyr68 phosphorylation of Chk2 kinase, a DNA damage response checkpoint kinase, following irradiation. However, CK2 inhibition did not decrease the ability of these glioma cells to repair double-strand DNA breaks, as assessed by COMET assays and γ-H2Ax staining. Likewise, apigenin and siRNA-induced depletion of CK2 failed to sensitize glioma cells to the cytotoxic effect of 2 to 10 G-rays of γ irradiation, as assessed by clonogenic assays. These results contrast with those found in other cancer types, and urge to prudence regarding the inclusion of malignant glioma patients in clinical trials that assess the radiosensitizing role of CK2 inhibitors in solid cancers.

HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation.

The human T-lymphotropic virus type 1 (HTLV-1) is efficiently transmitted through cellular contacts. While the molecular mechanisms of viral cell-to-cell propagation have been extensively studied in vitro, those facilitating the encounter between infected and target cells remain unknown. In this study, we demonstrate that HTLV-1-infected CD4 T cells secrete a potent chemoattractant, leukotriene B4 (LTB4). LTB4 secretion is dependent on Tax-induced transactivation of the pla2g4c gene, which encodes the cytosolic phospholipase A2 gamma. Inhibition of LTB4 secretion or LTB4 receptor knockdown on target cells reduces T-cell recruitment, cellular contact formation and virus propagation in vitro. Finally, blocking the synthesis of LTB4 in a humanized mouse model of HTLV-1 infection significantly reduces proviral load. This results from a decrease in the number of infected clones while their expansion is not impaired. This study shows the critical role of LTB4 secretion in HTLV-1 transmission both in vitro and in vivo.

Monitoring molecular response in adult T-cell leukemia by high-throughput sequencing analysis of HTLV-1 clonality

Monitoring molecular response in adult T-cell leukemia by high-throughput sequencing analysis of HTLV-1 clonality

Identification and characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts reveals their constitutive expression in leukemic and pre-leukemic clones

Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells and produces a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about ∽5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The 5’LTRs of both the BLV and HTLV-1 proviruses are transcriptionally silent in tumors, however they are not entirely quiescent, with the HLTV-1 antisense transcript HBZ and the BLV microRNAs constitutively expressed in tumors. Here, using RNA-seq, we demonstrate that in addition to microRNAs, the BLV provirus also constitutively expresses two antisense transcripts in all BLV infected samples examined. The first transcript (AS1) has alternate potential polyadenylation sites generating a short transcript of ∽600bp (aS1-S) and a less abundant longer transcript of ∽2200bp (AS1-L). Alternative splicing also creates a second transcript of ∽400bp (AS2) utilizing the first exon of AS1. Production of AS transcripts from the 3’LTR was supported by reporter assays demonstrating that the BLV LTR has substantial and Tax-independent antisense promoter activity. BLV AS transcripts predominantly localize in the nucleus. Examination of protein coding potential showed AS2 to be non-coding, while the AS1-S/L transcripts coding potential is ambiguous, with a small potential open reading frame (ORF) of 264bp present. The AS1-L transcript overlaps the BLV microRNAs transcribed in the sense direction. Using high throughput sequencing of RNA-ligase-mediated (RLM) 5’ RACE products, we show that the perfect complementary between the transcripts leads to RNA-induced silencing complex (RISC) mediated cleavage of AS1-L. Furthermore, experiments using BLV proviruses where the microRNAs were removed or inverted point to additional transcriptional interactions between the two viral RNA species. Knock down of AS1-S/L using locked nucleic acids (LNAs) showed no obvious effect on the cells phenotype. While a detailed elucidation of the BLV antisense transcripts function remains in the future, the constitutive expression in all samples examined, points to a vital role for the transcripts in the life cycle and oncogenic potential of BLV.

Improving the methodology for the detection of proviral integration sites in the host genome via high throughput sequencing

Bovine Leukemia Virus (BLV) and the closely related Human T-cell leukemia virus-1 (HTLV-1) are deltaretrovirus that induce leukemia/lymphoma in about ~5% of infected individuals. The mechanisms responsible for cellular transformation have remained largely enigmatic as both viruses are largely transcriptionally silent in tumors and show multiple integration sites in the host genome. The recent application of high throughput sequencing to track proviral insertion sites in the host genome has provided a number of insights into the evolution of deltaretrovirus infections and the progression of tumor clones in deltaretrovirus induced leukemia/lymphoma. However the protocols currently utilised have a number of limitations, including relatively high sequencing costs, the use of custom sequencing primers, no examination of the region upstream of the provirus and limited dynamic range for determining clone abundance. We have developed an alternative high throughput sequencing protocol for identifying proviral integration sites in BLV and HTLV-1 infected individuals that uses off-the-shelf Illumina primers for the addition of adapters and indexes. This greatly simplifies the process of multiplexing libraries and does away with the need for custom sequencing primers. Additionally our approach assays the region upstream of the provirus in addition to the downstream region, giving additional information on the frequency of 5’ deletions in proviruses and increasing the dynamic range of the assay. We have tested the approach on over 1 BLV and HTLV-1 samples, representing both tumors and preleukemic stages. Our approach allowed for a more accurate determination of clone abundance in tumors and by assaying the 5’ end of the provirus identified clones overlooked with previously published methods. Finally, by facilitating greater multiplexing of libraries we have reduced the cost to a level where the technique may be attractive in a clinical setting.