Role of the MLL-AF4 chimeric protein in the molecular pathogenesis of t(4;11) acute lymphoblastic leukemia

Chromosomal rearrangements involving the Mixed Lineage Leukemia (MLL) gene are associated with very aggressive forms of acute lymphoblastic leukemia (ALL), often refractory to conventional therapies. In particular, patients carrying the translocation t(4;11)(q21;q23) have the worst prognosis among patients with other MLL-associated malignancies. Although it has been largely shown that the MLL-AF4 fusion protein has the capability to up-regulate genes involved in the self-renewal/differentiation balance of the hematopoietic stem cell, the mechanism induced by this oncoprotein is still poorly understood. Previous functional proteomic studies performed in our group identified the molecular partners of the native AF4 protein, the most common MLL translocation partner in infant ALL, and confirmed that this protein is deeply involved in a complex protein network, important for the regulation of the Pol II-dependent transcription. In this study, we cloned in an eukaryotic expression vector the complete cDNA encoding MLL-AF4 and transiently expressed the recombinant protein in Hek293 cells. In order to understand which molecules take part in the aberrant pathway induced by the MLL-AF4 oncoprotein, we aimed to identify some of its molecular interactors, starting from the proteins that are already known to interact with AF4. We showed that MLL-AF4 binds to CdK9 that by interacting with cyclin T1 forms the positive elongation factor (P-TEFb), which is involved in the activation of the Pol II elongation machinery. We also found that MLL-AF4 interacts with CRSP130 and CRSP33, two members of the so-called “Mediator Complex”, thus suggesting that MLL-AF4 is involved in the regulation of the Pol II-dependent transcription. Interestingly, we found that MLL-AF4 also interacts with the tyrosine-kinase receptor FGFR2, and with a protein belonging to the family of 14-3-3s (the isoform θ), involved in diverse intracellular pathways. Moreover, we observed down-regulation in the expression of HoxA9, one of the MLL target genes, in the cells co-expressing both recombinant MLL-AF4 and 14-3-3 θ, thus suggesting that this interaction could modulate transcriptional processes induced by MLL-AF4. Elucidating the role of fusion protein interactors such as FGFR2 and 14-3-3 θ is very important for identifying new molecular targets for the therapy of the MLL-AF4-dependent B-cell ALL

Automodified Poly(ADP-Ribose) Polymerase Analysis to Monitor DNA Damage in Peripheral Lymphocytes of Floricoltorists Occupationally Exposed to Pesticides

Background:Increased DNA damage and the propension to cancer development, depend on the modulation of the mechanisms to control and maintain genomic integrity. Poly(ADP-Ribose)Polymerase activation and automodification are early responses to genotoxic stress. Upon binding to DNA strand breaks, the enzyme, a molecular DNA nick sensor,is hyperactivated: this is the first step in a series of events leading to either DNA repair or apoptosis. Enzyme hyperactivation and automodification can be easily measured and are widely used to look at DNA damage extent in the cell. We investigated whetherthese two markers (increasedcatalytic activity and auto modification), could help to monitor DNA damage in lymphocytes of flower growers from Southern Italy, occupationally exposed to pesticides. Methods: Peripheral lymphocyte lysates were analysed for Poly(ADP-Ribose) Polymerase activity, and by SDS-PAGE and anti-Poly(ADP-Ribose)Polymerase 1-antibody to measure automodified Poly(ADP-Ribose) Polymerase levels by densitometry. Results:Poly(ADP-Ribose)Polymerase activity levels were consistent with those of enzyme auto-modification. Growers daily exposed to pesticides, showed both biomarkers very high, either in the presence or in the absence of pathologies. Conclusions:PARP activity and auto-modification in peripheral blood lymphocytes are possible, non-invasive, and routinar tools to monitor the healthy conditions of floricoltorists

From generic gastro-enteric diseases to cancer: a family case study by molecular approaches.

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Prospective Isolation and Characterization of Genetically and Functionally Distinct AML Subclones

Intra-tumor heterogeneity caused by clonal evolution is a major problem in cancer treatment. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia (AML) samples. We identified 50 leukemia-enriched plasma membrane proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth, and engraftment behavior, as determined by RNA sequencing, DNase I hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture, and xenograft transplantation. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment