The spliceosome: a novel multi-faceted target for therapy.

The spliceosome is a dynamic and flexible ribonucleoprotein enzyme that removes intronic sequences in a regulated manner. Spliceosome action enables one stretch of genomic DNA sequence to yield several mRNAs that encode different proteins. It depends on a flexible mechanism for selecting splice sites, which calls for regulatory sequences (splicing enhancers or silencers) recognized by cognate trans-acting protein factors and constitutive ribonucleoprotein devices to build up the catalytic core. The identification of both types of elements now offers a comprehensive insight into how the spliceosome is adapted to carry out the removal of different introns and suggests novel therapeutic targets to, ultimately, restore a physiological pattern of alternatively spliced variants in a large repertoire of pathologies.

Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance.

The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16) that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

[Alternative splicing: a novel pharmacological target with wide therapeutic potential]. / L'epissage alternatif: une nouvelle cible pharmacologique aux potentialités thérapeutiques très larges.

Alternative splicing is a process by which a single stretch of genomic DNA yields several mRNAs encoding different proteins. Once believed to be a marginal phenomenon, alternative splicing now appears to be widespread among higher organisms and to be behind a large repertoire of human diseases. It involves a flexible mechanism for selecting splice sites, based on regulatory sequences recognized by cognate trans-acting protein factors (stimulatory SR proteins, or their antagonists). This RNA-protein interaction provides two types of targets for therapeutic manipulation. Masking regulatory RNA sequences with an antisense strategy is the most obvious, and encouraging results are beginning to accrue. Our lab is currently developing an entirely new approach in which activating proteins are targeted by small chemical molecules. A large screening program has been conducted with the chemical library from the Curie Institute. Several molecules (all indole derivatives) were found to counter the stimulatory effects of individual activating proteins, and have been selected for further development.

Spontaneous apoptosis in the intra-ductal component's stroma of breast invasive carcinoma.

Spontaneous apoptosis by in situ detection of DNA fragmentation (DNAf) was investigated in breast invasive ductal carcinoma (IDC) frozen samples removed from 61 untreated patients. The incidence of DNAf was low in carcinoma cells and was mainly detected in the stroma. In the stroma at a distance from carcinoma cells, DNAf was inversely related to estradiol plasma level variations (p=0.01), indicating that it probably remained under physiological hormonal regulation. In the stroma adjacent to carcinoma cells, DNAf was correlated to tumor progression parameters such as the presence of a comedo intra ductal carcinoma (DCIS) component (p=0.001) and axillary lymph node metastasis (p=0.002), suggesting that this stromal compartment more probably represented a tumoral component closely associated to epithelial tumor cells. Therefore, the detection of DNAf in the adjacent stroma of breast carcinoma could help to predict progression in non invasive tumors and also in invasive tumors in those patients without lymph node invasion.

[From genomics to post-genomics: back to the origins?]. / De la génomique à la post-génomique: un retour aux origines?

The purpose of this review is to emphasize the multiplicity and importance, physiological and pathological, of gene regulation levels which operate after the initiation step of transcription. Albeit crucial, this step is only the first one of a long cascade of events which eventually end up in the selection of functional messengers appropriate to the nature of the cells and to their immediate needs. Throughout this long pathway, of which only a few steps will be mentioned here, this review will attempt to address the central role played by RNA, not only as a substrate but as an actor of its own regulation. Emphasis will be put on the numerous connections with pathology up to the development of new therapeutics specifically targeting RNA. It highlights the perennity of basic questions, nearly half a century old, for which genomics, short of new concepts, will provide the databases and tools required to access to the immense field of functional genomics, the only one likely to bring relevant answers for therapeutics.

[RNA and cancer]. / ARN et cancer.

The aim of this review is to draw the attention to the numerous steps of gene expression which operate at the RNA level and which are significant drivers of the transformation process. The analysis of genomic abnormalities was at first limited to gross chromosomal alterations and to DNA point mutations. Then came the era of transciptomes which were originally believed, since they were mRNAs, to be a faithful reflection of the expressed proteins. As a matter of fact, this first-generation transcriptomics gave only a global, quantitative, assessment of gene expression at a given locus but overlooked the qualitative diversity of the messenger population generated by alternative splicing. We will show that beyond their essential role in the regulation of functions specific to metazoan like development, alternative splicing brings about an important vulnerability to mutations which is at the origin of many pathologies including cancer. The second aspect covered by this review is that of a rather novel category of RNAs, the microRNAs which, although non-coding, functionally behave as oncogenes or tumor suppressors through the negative control they exert on conventional oncogenes or suppressors. Beyond their diagnostic and prognostic interest, these two mechanisms offer entirely novel potential therapeutic targets.

[Genetic predisposition to prostate cancer]. / La prédisposition génétique au cancer de la prostate.

The advent and recent use of Genome-Wide Association studies (GWAS) for the search of genetic predisposition markers for prostate cancer since 2006 has put a very strong emphasis on the 8q24 locus where several single nucleotide polymorphisms (SNP) have been significantly associated with an increased relative risk. A wealth of recent papers have all confirmed the interest of this locus and identified several others. Interestingly, these markers seem to have additive effects pointing to the high complexity of prostate cancer predisposition. This situation along with our current inability to identify any causal gene(s) in these regions make these findings difficult to translate into routine clinical practice at the present time, a fortiori in terms of population screening.

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors.

The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have used in vitro splicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction. Importantly, compounds of this family specifically inhibit exonic splicing enhancer (ESE)-dependent splicing, because they interact directly and selectively with members of the serine-arginine-rich protein family. Treatment of cells expressing reporter constructs with ESE sequences demonstrated that selected indole derivatives mediate inhibition of ESE usage in vivo and prevent early splicing events required for HIV replication. This discovery opens the exciting possibility of a causal pharmacological treatment of aberrant splicing in human genetic disorders and development of new antiviral therapeutic approaches.