Anti-leukemic activity of microRNA-26a in a chronic lymphocytic leukemia mouse model.

Dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). The Eµ-TCL1 transgenic mouse develops a form of leukemia that is similar to the aggressive type of human B-CLL, and this valuable model has been widely used for testing novel therapeutic approaches. Here, we adopted this model to investigate the potential effects of miR-26a, miR-130an and antimiR-155 in CLL therapy. Improved delivery of miRNA molecules into CLL cells was obtained by developing a novel system based on lipid nanoparticles conjugated with an anti-CD38 monoclonal antibody. This methodology has proven to be highly effective in delivering miRNA molecules into leukemic cells. Short- and long-term experiments showed that miR-26a, miR-130a and anti-miR-155 increased apoptosis after in vitro and in vivo treatment. Of this miRNA panel, miR-26a was the most effective in reducing leukemic cell expansion. Following long-term treatment, apoptosis was readily detectable by analyzing cleavage of PARP and caspase-7. These effects could be directly attributed to miR-26a, as confirmed by significant downregulation of its proven targets, namely cyclin-dependent kinase 6 and Mcl1. The results of this study are relevant to two distinct areas. The first is related to the design of a technical strategy and to the selection of CD38 as a molecular target on CLL cells, both consenting efficient and specific intracellular transfer of miRNA. The original scientific finding inferred from the above approach is that miR-26a can elicit in vivo anti-leukemic activities mediated by increased apoptosis.

Ultrastructure of internal jugular vein defective valves.

OBJECTIVES: To study the ultrastructure of intraluminal defects found in the internal jugular vein by using a scanning electron microscopy. METHODS: Using a scanning electron microscopy, intraluminal septa and/or defective valves blocking the flow in the distal internal jugular vein of seven patients were studied together with the adjacent wall and compared with control specimen. RESULTS: The internal jugular veins' wall showed a significant derangement of the endothelial layer as compared to controls. Surprisingly, no endothelial cells were found in the defective cusps, and the surface of the structure is covered by a fibro-reticular lamina. CONCLUSIONS: Although the lack of endothelial cells in the internal jugular vein intraluminal obstacles is a further abnormality found in course of chronic cerebrospinal venous insufficiency, our investigation cannot clarify whether this finding is primary or caused by progressive loss of endothelium in relation to altered haemodynamic forces and/or to a past post-thrombotic/inflammatory remodelling.

Nanoparticles loaded with Nutlin-3 display cytotoxicity towards p53(wild-type) JVM-2 but not towards p53(mutated) BJAB leukemic cells.

The small molecule Nutlin-3 is a potent antagonist of the murine double minute 2 (MDM2)/p53 interaction exhibiting promising therapeutic anti-cancer activity. Nutlin-3 has been proposed as an anti-neoplastic agent for the treatment of onco-hematological diseases characterized by a lower incidence of p53 mutation with respect to solid tumors. Indeed, based on its selective non-genotoxic p53 activation, Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy. To overcome the poor bioavailability of Nutlin-3, we have assessed the potential efficacy of Nutlin-3 loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) against hematological malignancies. To test the specificity of the anti-leukemic activity, we have used leukemic cell lines characterized by different p53 status (JVM-2 and BJAB). NP loaded with Nutlin-3 (NP-Nutlin) were rapidly taken up by the leukemic cells and were as effective as native Nutlin-3 in promoting both induction of apoptosis and cell cycle arrest in p53(wild-type) JVM-2 cells, but not in p53(mutated) BJAB cells. Moreover, injection of NP-Nutlin, but not of free Nutlin-3, in a JVM-2-derived xenograft mouse model, reduced the subcutaneous tumor volume and promoted induction of apoptosis in the tumor mass. Overall, the chemical and structural characteristics of the NP-Nutlin-3, as well as their biological activity in vitro and in vivo, made them promising for further preclinical evaluations as potentially useful anti-leukemic carriers.

MDM2 non-genotoxic inhibitors as innovative therapeutic approaches for the treatment of pediatric malignancies.

Since the discovery of p53 as "guardian of the genome", a large number of efforts have been put in place in order to find molecular strategies aiming to restore p53 wild-type functions, particularly in the light of the fact that its pathway results ineffective in most tumors even though they have non-mutated p53. In this context, pediatric cancers, that are mostly p53 wild-type at the time of diagnosis, represent an ideal target for such therapeutic approach. Within the several mechanisms and proteins ruling p53 activity, the murine double minute 2 (MDM2) is its crucial negative regulator, frequently found overexpressed in p53-wild-type tumors. The development of new technologies such as nuclear magnetic resonance structure analyses, computational structure-based design studies, and library peptides screening have recently led to the discovery and characterization of a large number of compounds belonging to different chemical families that are able to target the interaction p53-MDM2, rescuing the p53 wild-type pathway with an overall pro-apoptotic and anticancer activity. Within the preclinical assessment of these molecules, the cis-imidazoline analogue Nutlin-3 has definitely attracted great interest for its in vitro and in vivo antitumor activity in several pediatric cancer models, either as single agent on in combination with standard chemotherapy. In this light, the aim of this review is to summarize the main preclinical evidences of the potential of MDM2 inhibitors for the treatment of childhood cancers and the key suggestions coming from their assessment in the treatment of adult cancers as proof of concept for future pediatric clinical studies.

State of the art of the therapeutic perspective of sorafenib against hematological malignancies.

The bi-aryl urea multi-kinase inhibitor Sorafenib (BAY 43-9006, Nexavar) was initially approved for the treatment of unresectable hepatocellular carcinoma and advanced renal cell carcinoma. Eleven years after its first description in PubMed, the therapeutic potential of Sorafenib has been evaluated in an increasing number of studies, mainly focused on solid tumors. More recently, the potential usefullness of Sorafenib has started to emerge also against hematological malignancies. At the molecular level, besides the RAF kinase pathway, which represents the first therapeutic target of Sorafenib, additional kinases, in particular the vascular endothelial growth factor receptor, have been identified as important targets of Sorafenib. A great interest for the potential use of Sorafenib against acute myeloid leukemia (AML) arose when it was demonstrated that a specific mutation of a kinase gene, called FMS-like tyrosin-kinase-3- internal tandem duplication (FLT-3-ITD) and occurring in more than 30% of AML, represents a molecular target of Sorafenib. However, recent phase I and II clinical studies showed that, in spite of its ability to suppress the activity of this mutated kinase, resistence to Sorafenib rapidly occurs in AML, suggesting that Sorafenib will be more effective in combined therapy than used as single drug. Another critical molecular target of Sorafenib is the anti-apoptotic protein Mcl-1. The ability of Sorafenib to rapidly shut-off Mcl-1 in virtually all the hematological malignancies investigated, including the B-chronic lymphocytic leukemia, represents a key element for its antileukemic activity as well as for therapeutic combinations based on Sorafenib. In this respect, it is of particular interest that many chemotherapeutic drugs or innovative anti-neoplastic compounds, such as recombinant TRAIL or inibitors of MDM2 protein, are either unable to down-regulate Mcl-1 or in some instances promote a paradoxical induction of Mcl-1. In this review, the growing evidences for the role of Mcl-1 in mediating the anti-leukemic activity of Sorafenib will be discussed in relationship with promising therapeutic perspectives.

Exposure of B cell chronic lymphocytic leukemia (B-CLL) cells to nutlin-3 induces a characteristic gene expression profile, which correlates with nutlin-3-mediated cytotoxicity.

By analyzing the cDNA obtained from 16 B-cell chronic lymphocytic leukemia (B-CLL) patient samples, we found that Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction, induced a characteristic gene expression profile (GEP) signature in 13 out of 16 B-CLL samples. The lack of Nutlin-3-induced GEP signature in 3 out of 16 B-CLL samples was not due to p53 deletion and/or mutation, as demonstrated by FISH analysis and p53 sequencing. Of note, the 3 B-CLL samples in which Nutlin-3 did not elicit the GEP signature were also less susceptible to Nutlin-3-mediated cytotoxicity with respect to the remaining 13 B-CLL samples. However, the partial lack of response in these p53 wild-type B-CLL samples was not due to defects in the ability of Nutlin-3 to promote p53 induction, as confirmed by the rapid accumulation of p53 protein at Western blot analysis in response to Nutlin-3 in all samples examined. Upon exposure to Nutlin-3, the genes up-regulated with the highest score in the majority of B-CLL cells were all known p53-target genes, including genes involved in apoptotic pathways, such as FAS and BAX, as well as MDM2. Taken together, our data indicate that the ability of Nutlin-3 to induce a characteristic GEP signature correlates with its cytotoxic potential in p53 wild-type B-CLL cells. However, in some p53 wild-type B-CLL samples, the response to Nutlin-3 cannot be predicted on the basis of FISH analysis or p53 sequencing.