Nicolò Musso

Clonal selection of 11q CN-LOH and CBL gene mutation in a serially studied patient during MDS progression to AML

By conventional metaphase and SNP array cytogenetics we serially studied a patient affected by high-risk myelodysplastic syndrome (MDS), documenting the conversion from partial trisomy 8q to trisomy 8 and partial tetrasomy 8q during progression to acute myeloid leukemia (AML). Moreover, the serial application of high resolution genomic array analysis at different disease stages allowed the description of cryptic abnormalities and the demonstration of their enrichment in the AML phase. In particular the detection and quantification of a copy-neutral loss of heterozygosity region located in chromosome 11q guided the search for point mutations in the CBL gene, thus allowing the escription of the novel missense mutation K382E and the demonstration of its selection during progression to secondary AML.

Broad copy neutral‐loss of heterozygosity regions and rare recurring copy number abnormalities in normal karyotype‐acute myeloid leukemia genomes

We analyzed, by the latest high‐resolution SNP arrays, 19 Normal Karyotype (NK)‐AML patients at diagnosis (Dx) and remission (R) phases, to determine the number of tumor‐associated copy number abnormalities (CNAs) and copy neutral‐loss of heterozygosity (CN‐LOH) regions per patient and to identify possible recurring genomic abnormalities. The number of tumor‐associated CNAs was determined after comparison of matched Dx/R samples using stringent conditions able to reduce the number of false positive CNAs. With the exception of a single outlier case, a low number of CNAs per patient was detected (median value of 1 somatic loss or gain per patient). However, a high prevalence of CNAs (60–70% of the patients showed at least one tumor‐associated gain or loss) and few recurring CNAs were observed, thus providing new hints towards identification of cooperating mutations. An extensive search of all tumor‐associated CN‐LOH regions >1 Mb revealed only three broad regions (terminal 12Mb of 22q, terminal 27Mb of 1p and the whole chromosome 21) in three patients out of 19 (16%). CN‐LOH of the whole chromosome 21 was responsible for homozygosity of a missense mutation (R80C) of RUNX1/AML1. Our study suggests that a relative submicroscopic copy number stability NK‐AML genomes is associated with low recurrence of specific CNAs and CN‐LOH in NK‐AML patient population. Sequencing of candidate genes in the identified CNAs and CN‐LOH regions should be considered a priority in the search of novel driver mutations of AML.

Modeling, design and synthesis of new heteroaryl ethylenes active against the MCF-7 breast cancer cell-line

A dataset of 50 compounds was used to generate a QSAR model and to design 9 new heteroaryl ethylenes. These compounds were synthesized, tested in vitro and a significant agreement with in silico predictions observed. Studies using Laser Scanning Confocal Microscopy pointed out that the compounds may act by different mechanisms.

Detailed Analysis of Apoptosis and Delayed Luminescence of Human Leukemia Jurkat T Cells after Proton Irradiation and Treatments with Oxidant Agents and Flavonoids

Following previous work, we investigated in more detail the relationship between apoptosis and delayed luminescence (DL) in human leukemia Jurkat T cells under a wide variety of treatments. We used menadione and hydrogen peroxide to induce oxidative stress and two flavonoids, quercetin, and epigallocatechin gallate, applied alone or in combination with menadione or H2O2. 62 MeV proton beams were used to irradiate cells under a uniform dose of 2 or 10 Gy, respectively. We assessed apoptosis, cell cycle distributions, and DL. Menadione, H2O2 and quercetin were potent inducers of apoptosis and DL inhibitors. Quercetin decreased clonogenic survival and the NAD(P)H level in a dose-dependent manner. Proton irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G2/M arrest. Quercetin reduced apoptosis and prolonged the G2/M arrest induced by radiation. DL spectroscopy indicated that proton irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus explaining the massive necrosis induced by 10 Gy of protons and also suggested an equivalent action of menadione and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within Complex I, probably the rotenone-binding site.

Resveratrol-Related Polymethoxystilbene Glycosides: Synthesis, Antiproliferative Activity, and Glycosidase Inhibition

A small library of polymethoxystilbene glycosides (20-25) related to the natural polyphenol resveratrol have been synthesized and subjected, together with their aglycones 17-19, to an antiproliferative activity bioassay toward Caco-2 and SH-SY5Y cancer cells. Six of the compounds exhibit antiproliferative activity against at least one cell line. In particular, compounds 17 and 18 proved highly active on at least one of the two cell cultures. Compound 18 showed a GI50 value of 3 μM against Caco-2 cells, a value comparable to that of the anticancer drug 5-fluorouracil. The closely related compound 19 proved inactive, and its conjugates 22 and 25 showed weak cell growth inhibition. The results indicate that minimal differences in the structure of both polymethoxystilbenes and their glycosides can substantially affect the antiproliferative activity. The possible hydrolytic release of the aglycones 17-19 by β-glucosidase or β-galactosidase was also evaluated. Compounds 20-25 were also tested as potential β-glucosidase, β-galactosidase, and α-glucosidase inhibitors. A promising inhibitory activity toward α-glucosidase was observed for 21 (IC50 = 78 μM) and 25 (IC50 = 70 μM), which might be indicative of their potential as lead compounds for development of antidiabetic or antiobesity agents.

Decreased expression of GRAF1/OPHN-1-L in the X-linked alpha thalassemia mental retardation syndrome

BackgroundATRX is a severe X-linked disorder characterized by mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassemia. The disease is caused by mutations in ATRX gene, which encodes a protein belonging to the SWI/SNF DNA helicase family, a group of proteins involved in the regulation of gene transcription at the chromatin level. In order to identify specific genes involved in the pathogenesis of the disease, we compared, by cDNA microarray, the expression levels of approximately 8500 transcripts between ATRX and normal males of comparable age.MethodscDNA microarray was performed using total RNA from peripheral blood mononuclear cells of ATRX and normal males. Microarray results were validated by quantitative real-time polymerase chain reaction.ResultscDNA microarray analysis showed that 35 genes had a lower expression (30-35% of controls) while 25 transcripts had a two-fold higher expression in comparison to controls. In the microarray results the probe for oligophrenin-1, a gene known for its involvement in mental retardation, showed a decreased hybridization signal. However, such gene was poorly expressed in blood mononuclear cells and its decrease was not confirmed in the quantitative real-time RT-PCR assay. On the other hand, the expression of an homologous gene, the GTPase regulator associated with the focal adhesion kinase 1/Oligophrenin-1-like (GRAF1/OPHN-1-L), was relatively high in blood mononuclear cells and significantly decreased in ATRX patients. The analysis of the expression pattern of the GRAF1/OPHN-1-L gene in human tissues and organs revealed the predominant brain expression of a novel splicing isoform, called variant-3.ConclusionsOur data support the hypothesis of a primary role for altered gene expression in ATRX syndrome and suggest that the GRAF1/OPHN-1-L gene might be involved in the pathogenesis of the mental retardation. Moreover a novel alternative splicing transcript of such gene, predominantly expressed in brain tissues, was identified.

Positive Caricature Transcriptomic Effects Associated with Broad Genomic Aberrations in Colorectal Cancer

We re-examined the correlation between Broad Genomic Aberrations (BGAs) and transcriptomic profiles in Colorectal Cancer (CRC). Two types of BGAs have been examined: Broad Copy-Number Abnormal regions (BCNAs), distinguished in gain- and loss-type, and Copy-Neutral Loss of Heterozygosities (CNLOHs). Transcripts are classified as “OverT” or “UnderT” if overexpressed or underexpressed comparing CRCs bearing a specific BGA to CRCs not bearing it and as “UpT” or “DownT” if upregulated or downregulated in cancer compared to normal tissue. BGA-associated effects were evaluated by changes in the “Chromosomal Distribution Index” (CDI) of different transcript classes. Data show that UpT are more sensitive than DownT to BCNA-associated gene dosage effects. “Over-UpT” genes are upregulated in cancer and further overexpressed by gene dosage, defining the so called “positive caricature transcriptomic effect”. When Over-UpT genes are ranked according to overexpression, top positions are occupied by genes implicated at the functional and therapeutic level in CRC. We show that cancer-upregulated transcripts are sensitive markers of BCNA-induced effects and suggest that analysis of positive caricature transcriptomic effects can provide clues toward the identification of BCNA-associated cancer driver genes.

Water soluble glucose derivative of thiocarbohydrazone acts as ionophore with cytotoxic effects on tumor cells

A novel water-soluble ionophore based on the thiocarbohydrazone moiety conjugated with glucose (GluTch) was synthesized through a simple two-step procedure. Structural elucidation was carried out in water solution by means of various spectroscopic techniques (NMR, UV-Vis, and CD), electrospray ionization mass spectrometry and density functional theory calculations. The flexible nature of the thiocarbohydrazone moiety of the new glycoderivative compound induced both different coordination motifs and stoichiometry towards copper and zinc. Cytotoxicity assays of the ligands on the human normal keratinocyte NCTC-2544, MDA-MB-231 breast cancer and PC-3 human prostate adenocarcinoma cell lines demonstrated that i) higher activity on cancer cells growth inhibition compared to a normal cell line; ii) the introduction of the glucose unit does not alter the cytotoxic activity of the underivatized ionophore ligand and iii) the presence of copper ion improves the activity of the thiocarbohydrazones.

Synthesis and Experimental Validation of New Designed Heterocyclic Compounds with Antiproliferative Activity versus Breast Cancer Cell Lines MCF-7 and MDA-MB-231

Recent drug discovery efforts are highly focused towards identification, design, and synthesis of small molecules as anticancer agents. With this aim, we recently designed and synthesized novel compounds with high efficacy and specificity for the treatment of breast tumors. Based on the obtained results, we constructed a Volsurf+ (VS+) model using a dataset of 59 compounds able to predict the in vitro antitumor activity against MCF-7 cancer cell line for new derivatives. In the present paper, in order to further verify the robustness of this model, we report the results of the projection of more than 150 known molecules and 9 newly synthesized compounds. We predict their activity versus MCF-7 cell line and experimentally verify the in silico results for some promising chosen molecules in two human breast cell lines, MCF-7 and MDA-MB-231.

Anti-proliferative effects of Cetuximab and Trastuzumab in colorectal cancer cell lines

Colon cancer is one of the most common human malignancies and a leading cause of death worldwide. In Europe around 250.000 new colon cases are diagnosed each year, accounting for around 9% of all the malignancies (Labianca R et al., 2010; Berrino F et al., 2007). Dysregulation of the signalling pathways induced through EGF receptors (ErbB/ HER receptors) by their over-expression or constitutive activation can promote tumor processes (Lurje G et al., 2009), including colorectal cancer. Therefore, the ErbB/HER receptor family with their most prominent members EGFR and HER-2 represents validated targets for anti-cancer therapy. Cetuximab and trastuzumab are two monoclonal antibodies approved for treating, respectively, metastatic colorectal and breast cancer. Because the monotherapy with cetuximab in metastatic colorectal is often insufficient (Cunningham D et al., 2004), it is useful to develop complementary therapeutic strategies to enhance antibody efficacy. A possible approach is co-administration of inhibitors, targeting multiple members of the EGF receptor family. In this study we examined the effect of cetuximab and trastuzumab in combination using two human colon cancer cell lines as a model. We observed that the two drugs had a cytostatic effect and inhibited the proliferation of both the cell lines in a time- and concentration-dependent manner. However, the combination had lower efficacy on one cell line than the other, with growth inhibition of 31% in the former and 49% in the latter. This result was associated to specific changes in cell cycle distribution, while no apoptosis was observed. Chromosome copy number heterogeneity and aneuploidy in tumoral cell lines have been reported (Pellestor F et al., 1999). Our data deriving from the cell cycle analysis confirmed the aneuploidy and polyploidy in our cellular models and are useful to explain cellular response to the combination. We used fluorescent in situ hybridisation analysis to evaluate EGFR and HER-2 gene amplification status. Both the tumour cell lines resulted in an abnormal copy number for the two genes resulting from aneuploidy (polisomy of chromosome 7 and 17) which is not responsible for the difference in sensitivity to cetuximab and trastuzumab between the two cell lines. In order to understand and to improve the pharmacological efficacy of cetuximab and trastuzumab combination, it will be useful to elucidate the molecular mechanisms involved in their activity. This will allow to develop novel and interesting approaches to cancer therapy.