Notre équipe a précédemment démontré que l'Interleukine (IL)-24 une cytokine de la famille de l'IL-10 a un effet cytostatique voire cytotoxique sur des cellules B normales ou leucémiques mises préalablement en cycle mais non sur des cellules quiescentes. L'IL-24 inhibe également la différenciation plasmocytaire des cellules B humaines du centre germinatif dans un modèle de différentiation in vitro. Nous avons utilisé ce modèle pour analyser pour la première fois le transcriptome de cellules B stimulées ou non par IL-24 au bout de 6 et 36 h. Plusieurs transcrits impliqués dans le métabolisme et la réplication de l'ADN sont inhibés précocement à l'exception d'IGF-1 qui est stimulé. L'IGF1 ayant été décrit comme une molécule de survie des cellules B ou pré-B, nous avons analysé son effet biologique et démontré qu'il a au contraire un rôle proapoptotique à doses physiologiques. En revanche, l'IL-24 induit l'expression plus tardive des gènes de la voie mitochondriale de la mort cellulaire programmée (MCP). Cet effet est également retrouvé dans des LLC « IgVH mutées » ou non mais avec une cinétique distincte des cellules B normales. Au total, dans des cellules activées au préalable, l'IL-24 induit séquentiellement un blocage précoce du cycle cellulaire suivi d'une apoptose. D'autres gènes potentiellement importants dans la synapse immune ainsi que dans la régulation de l'immunité innée sont décrits et suggèrent que l'IL-24 a un rôle immunologique particulier au-delà de son effet cytostatique et potentiellement anti-tumoral / We have previously shown that Interleukin(IL)-24 a class-II cytokine of the IL-10 family has cytostatic and cytotoxic properties on normal and malignant human B-cells previously engaged into the cell cycle, but not on quiescent B-cells. IL-24 also inhibits the differentiation of germinal center B-cells in plasma cells in an in vitro model; the later was used to compare for the first time the transcriptome of B-cells cultured or not with IL-24 for 6 and 36h. Several "early" transcripts involved in DNA metabolism and replication were inhibited whereas that of Igf1 a molecule described as a B-cell growth factor was induced. We show herein that IgF1 has instead a proapoptotic role on B-cells at physiological concentrations. In contrast, several genes of the intrinsic apoptotic pathway were stimulated after 36h. This expression pattern was also found in CLL cells whether they were "IgVH mutated" or "unmutated", albeit with distinct kinetics from normal B-cells. In addition several genes belonging to the immune synapse and innate immunity were regulated by IL-24. These results disclose additional, possibly immunoregulatory properties, for IL-24 than its already described cytostatic and potentially anti-tumoral effects

Glioblastoma multiforme (GBM) is an aggressive cancer that affects millions of patients per year. Conventional therapies combining chemotherapeutic agents with radiation can only extend survival by a few months; therefore, there is a dire need for an effective means of treating this deadly disease. Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), currently in the early stages of FDA pre-IND drug trials, has proven to be an effective cancer specific cytokine, able to trigger the onset of mitochondrial dysfunction and/or autophagy. GBM’s have mutations that often result in the activation of cytoprotective cell signaling pathways, preventing cancer therapeutics and even MDA-7/IL-24 treatments from being effective. Since the discovery of MDA-7/IL-24 a number of groups have shown toxic effects in a variety of tumor cells. However, the lethality of MDA-7/IL-24 is not enough to eradicate the tumor. We hypothesized two xxiii rationales for this minimalistic effect. First, the MDA-7/IL-24 gene delivery mechanisms are not efficient or second, active pro-survival pathways are playing a role in protection. Here we have shown that the inhibition of cytoprotective cell-signaling pathways using small molecule inhibitors of mitogen-activated extracellular regulated kinase (MEK)1/2 and phosphatidyl inositol 3-kinase (PI3K) or AKT; mammalian target of rapamycin (mTOR) and MEK1/2; HSP90 inhibitor 17AAG; and the autophagy-inducing drug OSU-03012 (AR-12), enhances the toxicity of MDA-7/IL-24. In addition, the use of a modified recombinant adenovirus comprised of the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, proved to be a more effective, CAR-independent means of infecting and killing GBM cells in vitro and in vivo when compared to Ad.5-mda-7. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.

Melanoma differentiation associated gene-7 (mda-7) was discovered in the Fisher laboratory by subtraction hybridization of temporally spaced subtracted cDNA libraries prepared from terminally differentiated human melanoma cells treated with human fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ), an approach called ‘differentiation induction subtraction hybridization’ (DISH). mda-7 is located in human chromosome 1q32–33 and based on sequence homology, chromosomal localization, and its functional properties, the mda-7 gene is now classified as a member of the IL-10 family of cytokines and named IL-24. The mda-7/IL-24 cDNA encodes a protein of 206-amino acids with a predicted size of ~24-kDa, which contains an interleukin (IL)-10 signature motif at amino acids 101–121 (SDAESCYLVHTLLEFYLKTVF) shared by other members of the IL-10 family of cytokines. Sequence analysis revealed the presence of a 49-amino acid signal peptide suggesting that the molecule could be cleaved and secreted. Expression of MDA-7/IL-24 protein was detected in cells of the immune system (mainly by expression in tissues associated with the immune system, such as spleen, thymus and PBMC) and normal human melanocytes. Of interest, a progressive loss of MDA-7/IL-24 expression during melanoma progression suggests an inverse relationship between MDA-7/IL-24 expression and the evolution of melanocytes to various stages of melanoma. mda-7/IL-24 induces growth suppression in human melanoma and other cancer cells, without affecting normal cells. Subsequent studies provided consistent evidence that ectopic expression of mda-7/IL-24 employing a replication incompetent adenovirus (Ad.mda-7) resulted in apoptosis induction and cell death in a wide variety of solid tumors including melanoma, malignant glioma, carcinomas of the breast, kidney, cervix, colorectum , liver, lung, ovary and prostate sparing normal cellular counterparts, i.e., such as normal melanocytes, astrocytes, fibroblasts, and mesothelial and epithelial cells. The in vitro antitumor activity of mda-7/IL-24 readily translated into the in vivo situation in animal models containing human breast, prostate, lung and colorectal carcinomas and in malignant glioma xenografts. Moreover, the ability of mda-7/IL-24 to induce a potent “bystander cancer-specific killing effect” provides an unprecedented opportunity to use this molecule to target for destruction not only primary tumors, but also metastases. Based on its profound cancer-selective tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a Phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant (44%) clinical activity. This review focuses on the recent enhancements in our understanding of the mode of action of mda-7/IL-24 and its potential applications as a unique and promising effective cytokine-based gene therapy for human cancers. The first chapter explored the efficacy of a tropism-modified Ad-based cancer gene therapy approach for eradicating low CAR colorectal cancer cells. We show that in low CAR human colorectal cancer cells (RKO), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficient than Ad.5 delivering mda-7 (Ad.5-mda-7) in expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis and inhibiting in vivo tumor growth in a nude mouse xenograft model. Additionally, our in vitro and in vivo data confirms that BI-97C1 (Sabutoclax) profoundly sensitizes mda-7/IL-24 mediated toxicity in colorectal cancer. Thus, Ad.5/3-mda-7, alone and/or in combination with BI-97C1 (Sabutoclax), might represent an improved and more effective therapeutic approach for colorectal and other cancers. In view of the essential roles of anti-apoptotic Bcl-2 family proteins in tumorigenesis and chemoresistance, efforts are focused on developing small molecule inhibitors of Bcl-2 family proteins as potential therapeutics for cancer. Unfortunately, due to the unique structure of Mcl-1 as compared with Bcl-2 and Bcl-xL, currently employed inhibitors, such as ABT-737 or its clinical counterpart, ABT-263, display limited affinity for Mcl-1. Using nuclear magnetic resonance (NMR) binding assays and computational docking studies, we have recently identified a series of new Apogossypol derivatives, compound 3 (BI-79D10) and compound 11 (BI-97C1), with pan-Bcl-2- inhibitory potency. BI-79D10 binds to Bcl- xL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively. BI-97C1 (Sabutoclax) is an optically pure individual Apogossypol derivative that retains all the properties of BI-79D10 along with superior in vitro and in vivo efficacy. Because Mcl-1 is over-expressed in the majority of PCs, we hypothesized that suppressing Mcl-1 by treating human PC cells with BI-97C1 (Sabutoclax) would sensitize them to mda-7/IL-24-mediated cytotoxicity. The second chapter study highlights the noteworthy potential of a combinatorial approach involving mda-7/IL-24, a broad-acting anticancer gene, and BI-97C1 (Sabutoclax), which targets Mcl-1, to sensitize PC to mda-7/IL-24-mediated cytotoxicity, thereby enhancing therapeutic efficacy. Our data suggests that treatment with the combination regimen of mda-7/IL-24 and BI-97C1 (Sabutoclax) induces autophagy that facilitates apoptosis in association with up regulation of NOXA, accumulation of Bim, and activation of Bax and Bak. Treatment with mda-7/IL-24 and BI-97C1 (Sabutoclax) inhibited the growth of PC xenografts and suppressed PC development in an immunocompetent transgenic mouse model of PC. The third chapter study explored the efficacy of a tropism-modified CRCA cancer gene therapy approach for eradicating low CAR prostate cancer cells. We showed that in low CAR PC3 cells Ad.5/3-CTV is more efficient than Ad.5-CTV in delivering transgene (mda-7/IL-24), infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor ‘bystander’ effect in a nude mouse human prostate cancer xenograft and suppressed PC development in an immunocompetent transgenic mouse model of PC model.

Melanoma differentiation associated gene 7/Interleukin-24 (MDA-7/IL-24) is a secreted cytokine which acts as a tumor suppressor. It is capable of selectively killing cancer cells, regardless of anatomic origin, while sparing normal cells. miRNAs are master regulators of gene expression that can play two roles in cancer: tumor-suppression and oncogenesis. We identified a number of miRNAs that are regulated by MDA-7/IL-24 using a PCR plate array containing probes for miRNAs known to play a role in prostate cancer. We independently validated the array with qRT-PCR to identify three miRNAs which are downregulated by MDA-7/IL-24 treatment in DU145, PC3, and PC3ML prostate cancer lines. These miRNAs were miR-125a, miR-145, and miR-23b. Their gene targets were identified using TargetScan and confirmed to be regulated in our prostate cancer model. NLRC5, KLF4, and KLF15, respectively, were upregulated after treatment with MDA-7/IL-24. We focused on NLRC5 as a novel target of MDA-7/IL-24, which plays a role in immune evasion by cancer cells. NLRC5 is upregulated following inhibition of miR-125a. It is not downregulated by overexpression of miR-125a which suggests that more than one miRNA may be acting to regulate its expression. Finally, we determined that miR-125a is downregulated by MDA-7 through DICER, an important processing enzyme for miRNA biogenesis.

The essence of this diploma thesis is the design and implementation of driver for Profinet bus coupler from Phoenix Contact, which use computer's network card for communication. The proposal builds on the knowledge gained from available literature and analysis of Profinet protocol.