Thèses sur le sujet « Ras protein inhibitors »

Because of their role in oncogenesis, inhibition of Ras proteins, particularly of their tumorigenic variants, represents today one of the principal strategies finalized to the obtainment of new antitumoral therapies. Among the different possible approaches, one of the most innovative and less explored is represented by the inhibition of this protein activation, key event for the explication of their biological activity, but also for the Ras-induced tumoral cell transformation. Objective of this thesis has been the development of new small molecules able to inhibit, at least partially (total inhibition in fact would result lethal for cell), Ras protein activation, in particular the GEFs-promoted GDP/GTP nucleotide exchange. Inhibitors able of inactivating Ras have been previously described by Schering-Plough. All these molecules contain a phenylhydroxylamino group that binds Ras in a region close to the nucleotide binding site and one aromatic group. Nevertheless, they present some negative characteristics that prevent their employment as potential drugs: (1) they are chemically unstable and (2) they are insoluble in water and in the most commonly used organic solvents. In order to obtain new more efficient inhibitors, we adopted the rational drug design strategy. Firstly, we studied the structure-activity relationship (SAR) of Schering-Plough compounds and of new molecules containing variants of their functional groups that we designed and synthesized. The data collected demonstrated that the phenylhydroxylamino group is an essential pharmacophore, while other positions are not so critical for the biological activity.Keeping in mind this, we prepared new compounds in which the phenylhydroxylamino moiety is supported on glycidic templates, in an attempt to try to take advantage of carbohydrate capability of orienting substituents in space, in this case in a suitable manner for the interaction with Ras proteins. In addition, the sugar portion can improve compound pharmacokinetic properties and decrease their toxicity. In this way, a new class of Ras inhibitors was obtained, their biological activity and the nature of their interaction with the molecular target were characterized.

Statins are widely used to treat hypercholesterolemia. Statins inhibit cholesterol biosynthesis, thereby activating genes involved in cholesterol homeostasis, which are under the control of the Sterol Regulatory Element (SRE). Statins also have cholesterol-independent beneficial cardiovascular effects mediated through the phosphoinositide 3-kinase (PI3-K) / Akt signaling pathway and by inhibition of protein prenylation. Because statins inhibit the synthesis of isoprenoids, they can act by inhibiting the small signaling GTPases Ras and Rho, which require post-translational prenylation to become membrane-anchored and functional. We showed that simvastatin-mediated inhibition of protein prenylation does not appear to play a role in activation of SRE transcriptional activity in HepG2 cells. We also found that when isoprenoids were replenished, basal phospho-Akt decreased, suggesting that inhibition of prenylation by simvastatin mediates Akt phosphorylation. Future studies will be needed to investigate the role that inhibition of protein prenylation plays in the activation of the PI3-K/Akt pathway by simvastatin.
Department of Biology

Dissulfeto isomerase proteica como a PDIA1 tem sido implicada na progressão do câncer, porém os mecanismos envolvidos ainda não foram claramente identificados. Previamente, nós demonstramos um importante efeito da PDIA1 induzindo a superexpressão da Nox1 NADPH oxidase, associada à geração de espécie reativas de oxigênio (ROS). Uma vez que a perda na regulação de ROS envolve o crescimento tumoral, nós propusemos que a PDIA1 atua como um mecanismo regulador proximal na produção de ROS em tumores. No presente estudo, nós focamos no câncer colorretal (CRC) com distintos efeitos na ativação de KRas. Resultados provenientes de bancos de dados de RNAsec e validação direta, indicam um significante aumento na expressão de PDIA1 em CRC com alta ativação constitutiva da Kras (HCT116) vs. ativação intermediária (HKE3) ou basal (Caco2). A PDIA1 sustenta a produção de superóxido dependente da Nox1 em CRC; entretanto, observamos pela primeira vez uma ação dupla da PDIA1 correlacionada ao nível de ativação da Ras: em células Caco2 e HKE3, experimentos de perda de função indicam que o PDIA1 sustenta a produção de superóxido dependente de Nox1; no entanto, em células HCT116, PDIA1 limita a produção de superóxido pela Nox1. Este comportamento da PDIA1 é associado ao aumento da expressão / atividade da Rac1. A transfecção do mutante constitutivamente ativo Rac1G12V em células HKE3 faz com que a PDIA1 se torne restritiva a produção de superóxido dependente de Nox1, paralelamente, em células HCT116 tratadas com inibidor da Rac1, PDIA1 se torna favorável à produção de superóxido. Um screening em importantes vias de sinalização celular em HKE3 mostrou que a perda de função da PDIA1 promove inativação da GSK3? em paralelo à diminuicão da ativacção de Stat3; em HCT116 em estado basal, GSK3beta é inativada enquanto Stat3 está ativa, já o silenciamento da PDIA1 não resulta em nenhum efeito adicional. As implicações funcionais do silenciamento da PDIA1 incluíram uma diminuição da proliferação e migração celular em HKE3, não detectável em HCT116. Além disso, a PDIA1 parece sustentar a transição epitélio-mesenquimal (EMT), uma vez que após o silenciamento da PDIA1, observamos um aumento da expressão da E-caderina em HKE3 e uma diminuição em HCT116. Assim, a superativação da Ras se associa a uma alteração no padrão de regulação da Nox1 pela PDIA1. A supressão do efeito regulador da PDIA1 pela Kras é provavelmente devido a uma ativação sustentada da Rac1. Portanto, PDIA1 pode exercer um papel redox-dependente adaptativo crucial relacionado à progressão tumoral
Protein disulfide isomerases such as PDIA1 have been implicated in cancer progression, but the underlying mechanisms are unclear. We showed previously important PDIA1 effects enabling vascular Nox1 NADPH oxidase expression and associated generation of reactive oxygen species (ROS). Since deregulated ROS production underlies tumor growth, we proposed that PDIA1 acts as an upstream regulatory mechanism of tumor-associated ROS production. We focused on colorectal cancer (CRC) with distinct levels of KRas activation. Our results from RNAseq databanks and direct validation indicate significant increase in PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) or basal (e.g. Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we observed for the first time a dual effect correlated with Ras level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production; however, in HCT116 cells, PDIA1 restricted Nox1-dependent superoxide production. This PDIA1 behavior in HCT116 is associated with increased Rac1 expression/activity. Transfection of Rac1G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide; accordingly, in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide production. Screening of cell signaling routes affected by PDIA1 silencing showed induced GSK3beta inactivation and parallel decrease of active Stat3 in HKE3 cells; in baseline HCT116 cells, GSK3beta was inactivated and Stat3 active, whereas PDIA1 silencing had no further effect. Functional implications of PDIA1 silencing included a decrease of cell proliferation and migration in HKE3, not detectable in HCT116 cells. Also, PDIA1 may support epithelial-mesenchymal transition (EMT), since after PDIA1 silencing, E-cadherin expression increased in HKE3 and decreased in HCT116. Thus, Ras overaction associates with a switched in PDIA1 pattern regulation of Nox1. Ras-induced PDIA1 bypass may involve direct Rac1 activation. Therefore, PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression

In recent years, great advances have been made in understanding the molecular causes of human disease, but our ability to exploit these discoveries for therapeutic benefit is frequently limited by the inability to make drugs that target the processes responsible. Many diseases can be linked to the aberrant activity of proteins, and while the development of inhibitors for enzymes and extracellular targets is often feasible, these proteins account for only a small fraction of all the proteins in cells. The remaining proteins are, in most cases, considered therapeutically intractable and are sometimes referred to as "undruggable." Many proteins, particularly in higher organisms, carry out their activity in part through interactions with other proteins and biomolecules. The ability to specifically disrupt these interactions could have great therapeutic benefit, as it may provide a means of targeting otherwise intractable processes. The focus of this dissertation is on the development and characterization of molecules that inhibit the interactions of an “undruggable” protein target, Ras, which is linked to both the initiation and progression of a wide array of human cancers. Our approach has been to use high-throughput screening, coupled with directed evolution, to identify and improve small proteins (peptides) that bind Ras and block its ability to engage the effector proteins necessary for its oncogenic activity. We report these efforts, along with a series of biochemical experiments aimed at characterizing the properties and binding mechanism of the peptides discovered in the screen. These peptides bind the three human Ras proteins with mid-to-low nanomolar affinity, and with high specificity for Ras proteins over their close family members. The peptides directly engage the Ras effector domain, and can block Ras from binding a canonical effector protein in the context of cancer cell lysates. Based on a series of observations, we hypothesize that the peptides bind Ras as head-to-tail homodimers, and report preliminary attempts to exploit this observation and identify peptides with improved affinity to Ras. Finally, we discuss the preliminary results from a conceptually related effort to identify peptide inhibitors of the Myc transcription factor, which is another protein heavily implicated in human cancer.

RKIP was initially discovered as an endogenous inhibitor of the ERK and NF-κB pathways,and was also shown to prolong the activation of GPCRs via inhibition of the GRK2 protein. Now increasing evidence has linked RKIP to a metastases suppressing and chemo-sensitising role in cancer cells.The chemo-sensitising effect of RKIP was investigated in a colon carcinoma cell line using a variety of chemotherapeutic agents from conventional agents to newer targeted therapies. Initial results suggested that role of RKIP in the modulation of chemotherapeutic drug response was at the level of apoptosis; there did not appear to be great observable effects in the cell proliferative response and the cell cycle distribution of the colon carcinoma cells after treatment with selected agents. Apoptosis modulation by RKIP occurred after treatment with doxorubicin, FasL, paclitaxel and TRAIL. TRAIL-treated colon carcinoma cells displayed increased cell death as the levels of RKIP within the cell were increased. In contrast, doxorubicin, FasL and paclitaxel-treated cells displayed a scaffold-like response as the levels of RKIP were increased in the cell; with WT RKIP-expressing cells being more sensitive to doxorubicin, FasL and paclitaxel-induced apoptosis than low or high RKIP-expressing colon carcinoma cells. There was no modulation of 5-FU, cisplatin and etoposide-induced apoptosis by RKIP. Indeed, these three agents did not appear to induce cell death in this colon carcinoma cell line. RKIP modulation of chemo-sensitivity has never been shown before in a colon carcinoma cell line and this is the first time that doxorubicin and FasL-induced apoptosis has been shown to be modulated by RKIP. Further, it is shown here, for the first time, that the modulation of chemotherapy-induced apoptosis by RKIP can change depending upon the cytotoxic drug employed as treatment. TRAIL and FasL, both members of the TNF super-family, were selected for further analysis due to the distinctive cell death responses observed as a consequence of the levels of RKIP within the cell. WT RKIP cells were sensitive to FasL treatment, and high RKIP cells were most sensitive to TRAIL administration. Increased sensitivity of high RKIP-expressing colon cells to TRAIL treatment appeared to involve up-regulation of the DR5 receptor; down-regulation of the anti-apoptotic molecule Bcl-xl; pIKK which activates the NF-κB pathway; and TRAF2 which has been shown to activate the NF-κB pathway. Whether RKIP directly interacts with these molecules is unknown however RKIP has been shown to bind upstream activators of the NF-κB pathway and another TRAF subtype TRAF6. YY1 expression was evident in the TRAIL-treated cells but the expression was unchanged as the levels of RKIP within the cell were altered. The FasL-treated cells also displayed decreased pIKK levels as the levels of RKIP were increased; it is possible that NF-κB was behaving as both pro- and anti-apoptotic within this cell line. Thus RKIP inhibition of the NF-κB pathway may have prevented FasL-induced apoptosis in the high RKIP-expressing colon carcinoma cells. The expression of TRAF6, which has been shown to bind RKIP, displayed a scaffold-like response with WT RKIP-expressing cells having the highest TRAF6 expression. This was also the case for the transcriptional regulator YY1, thus it is possible that both YY1 and TRAF6 were behaving in a pro-apoptotic-like manner in the WT RKIP-expressing cells. TRAF2 was also evident in the FasL-administered cells but the expression did not change regardless of the levels of RKIP within the cell. Overall, it appears that differential expression of TRAF adaptor proteins is responsible for the contrasting responses of TRAIL and FasL-treated cells with low, WT and high RKIP expression. Utilisation of particular TRAF adaptors or TRAF combinations by the TRAIL and Fas receptors may also account for the pro- and anti-apoptotic roles of the NF-κB pathway, and the recruitment or down-regulation of other proteins dependent upon the cell stimulus. How RKIP affects these proteins requires further investigation, however these results are exciting and novel, and strengthen evidence surrounding the role of RKIP in chemosensitivity. On another note, RKIP has been shown to bind the PDE5 inhibitor PF-3717842, therefore investigation of the effects of the PDE5 inhibitors sildenafil citrate and vardenafil citrate on RKIP inhibition of the ERK pathway in a colon carcinoma cell line were examined. The effects of the PDE5 inhibitors were compared to the cell migration inhibitor locostatin that has been shown to bind and inhibit RKIP, and prevent the RKIP-Raf-1 interaction. With TPA and EGF stimulation, locostatin appeared to act in a manner consistent with its known function as an RKIP inhibitor. The PDE5 inhibitors sildenafil citrate and vardenafil citrate displayed a similar trend to that of locostatin, although their effects on the ERK pathway were not as potent. It is possible that after EGF stimulation, the strong activation of B-Raf was over-shadowing the subtle effects of the drug treatments. Under growth conditions, the RKIP inhibitor locostatin did not appear to behave as an inhibitor of RKIP nor did the PDE5 inhibitors sildenafil citrate and vardenafil citrate. It is possible that the strong activation of various growth and proliferative cascades was impinging upon the ERK pathway, were overshadowing the drug effects, or resulting in off-target (RKIP-unrelated) effects of the drugs. In summary, the role of RKIP within the cell is becoming an increasingly exciting avenue of research and is consistently yielding new and interesting roles and interactions within the cell. Understanding and elucidating the roles of this intriguing protein within the cell will not only strengthen our knowledge of signal transduction regulation and modulation, but may also provide a new source of targeted therapy and means of manipulation in the treatment of cancer and chemotherapeutic drug resistance.

Deregulation of kinase activity has emerged as a major mechanism by which cancer cells evade normal physiological functions such as growth and survival. The identification of mutations in FGFR-3 within non invasive tumours of bladder cancer and over expression of this receptor in invasive tumours and superficial tumours makes FGFR-3 a promising target in developing a therapy for the treatment of urothelial bladder cancer. It was found that inhibiting VEGFR-2 together with FGFR provides an attractive strategy for the development of new anti-angiogenic agents as a potential anti -cancer therapy. In this project, the three dimensional structure of both the FGFR and VEGFR-2 was used in conjunction with the de novo methods such as SPROUT to generate oxindole- based novel inhibitors of FGFR and VEGFR-2 with ICso values in the range of 1-10 flM (Compound 4.7 inhibits FGFR and VEGFR-2 with ICso values of 3.9 uM and 1.6 flM for respectively (Chapter 4). These compounds show encouraging anti-angiogenic activity in the lower u M concentrations. In Chapter 5, intramolecular H-bonding concept was used to design pyrazole-based inhibitors of FGFR and VEGFR-2 with 29% and 63% inhibition at 10 uM respectively. Benzo-fused designs based on pyrazole scaffolds yielded indazole based compounds with inhibitory effects of 45%, 41 % and 74% for FGFR-1, FGFR-3 and VEGFR-2 respectively. Shape similarity was used to find novel hinge binders replacing the indazole core with hydroxy-quinolinone moiety which showed an encouraging level of inhibition with 30% and 64% for FGFR and VEGFR-2 respectively. Purine based inhibitors were designed to exploit the smaller gatekeeper residue in both the FGFR and VEGFR-2, but all the compounds show weaker or no inhibition at 10 u M (Chapter 5). In Chapter 6, structure guided method were used to design oxadiazole-based novel VEGFR-2 inhibitor with an ICso value of 1.6 j.1M. All these approaches are novel used in complementary to HTS. In Chapter 3, SPROUT was used to design novel inhibitors of Ras protein and the preliminary analysis of these inhibitors show preferential binding of these inhibitors to the active form compared to the inactive form. These molecules were first amongst the de novo/designed inhibitors of Ras which could serve as a starting point f-or the future potential inhibitors of protein-protein interaction.

I leiomiomi uterini (detti anche fibromi, miomi) sono tumori benigni che originano dallo strato muscolare dell’utero (miometrio) e rappresentano la principale indicazione dell’isterectomia nel mondo. I leiomiomi uterini colpiscono circa il 77% delle donne in eta fertile e circa il 25% di esse presenta tumori con sintomatologia clinica evidente, tra cui la presenza di forte o anomalo sanguinamento uterino, dolore o pressione pelvica, infertilità e aborti ricorrenti. È comunemente noto che questi tumori sono caratterizzati da una elevata proliferazione cellulare ed una eccessiva deposizione di matrice extracellulare (ECM). Si ritiene che la crescita dei leiomiomi dipenda dall’azione degli ormoni ovarici mediante elementi intermedi come citochine e fattori di crescita. La Proteina Inibitore della Raf Chinasi (RKIP) ha un ruolo emergente come regolatore in diversi pathway molecolari ed è associato a un numero crescente di malattie, essendo coinvolto indiverse vie di trasduzione del segnale. Lo scopo della presente tesi è stato quello di indagare la presenza e il ruolo dell’RKIP nel leiomioma. Abbiamo dimostrato che l’RKIP è espresso nel miometrio e nel leiomioma. Per individuare il ruolo dell’RKIP, abbiamo eseguito esperimenti in vitro con un composto chimico quale la locostatina, capace di legarsi all’RKIP bloccandolo. Abbiamo dimostrato che il trattamento con la locostatina porta all’attivazione della via di segnale MAPK (fosforilazione di ERK), fornendo una opportuna validazione dell’efficacia nel bloccare l’RKIP. Inoltre, abbiamo dimostrato che l'inibizione dell’RKIP con la locostatina riduce le componenti della ECM, tra cui il collagene 1A1, la fibronectina, e il versican. In aggiunta, l'inibizione dell’RKIP con la locostatina riduce la proliferazione cellulare e la migrazione sia nelle cellule miometriali che di leiomioma. Infine, abbiamo dimostrato che il trattamento con la locostatina riduce l’espressione del GSK3β. Pertanto, anche se l'attivazione delle MAPK dovrebbe far aumentare la proliferazione e la migrazione, la destabilizzazione e l’inattivazione del GSK3β porta alla riduzione della proliferazione e della migrazione delle cellule miometriali e di leiomioma.
Uterine leiomyomas (fibroids, myomas) are benign (non-cancerous) tumors that origin from the smooth muscle layer of the uterus (myometrium), and are the most common indication for hysterectomy in the world. Uterine leiomyomas affect about 77% of women of reproductive-age, and approximately 25% of them bear clinically apparent tumors with symptoms like heavy or abnormal uterine bleeding, pelvic pain or pressure, infertility, and recurrent pregnancy loss. It is commonly known that these tumors are characterized by increased cell proliferation and excessive deposition of extracellular matrix (ECM). Growth of leiomyoma is thought to be dependent on ovarian hormones activity through intermediate elements such as cytokines and growth factors. Raf Kinase Protein Inhibitor (RKIP) has emerging roles as regulator of multiple signaling networks and is associated with an increasing number of diseases through its involvement with signal transduction pathways. The aim of the present thesis was to investigate the presence and the role of RKIP in leiomyoma. We demonstrated that RKIP is expressed in human myometrial and leiomyoma tissue. In order to define the RKIP role, we performed in vitro experiments with the chemical compound locostatin, known to bind and block RKIP. We showed that locostatin treatment results in the activation of the MAPK signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, we showed that RKIP inhibition by locostatin reduces ECM components, including collagen1A1, fibronectin, and versican. Moreover, the inhibition of RKIP by locostatin impairs cell proliferation and migration in both leiomyoma and myometrial cells. Finally, we demonstrated that locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization and inactivation of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells.

Thesis (Ph.D.)--Boston University
Metastatic melanoma is the major cause of skin cancer death, and the annual incidence of melanoma continues to increase. Despite the impressively high rates of response to BRAF inhibitors in patients with melanomas harboring BRAF mutations, most of these patients eventually relapse after developing resistance to the drug, due in part to secondary mutations in NRAS. Although NRAS mutation is the second most common genetic mutation in melanoma patients (after BRAF mutation), there is currently no treatment option that targets NRAS-mutated melanomas. Previous reports have demonstrated the sensitivity of cancer cell lines carrying RAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCδ), suggesting the possible association between RAS mutational status and sensitivity to PKCδ inhibition. I therefore hypothesized that PKCδ inhibitors might also be cytotoxic in melanomas with primary or acquired NRAS mutations. In this project, the effect of PKCδ inhibition, and the efficacy of a new PKCcS inhibitor, BJE6-106 (B106), in melanoma were investigated. Inhibition of PKCδ inhibited the growth of multiple human melanoma cell lines carrying NRAS mutations, and induced apoptosis mediated by terminal caspase activation. Analysis of the molecular mechanisms demonstrated activation of the JNK pathway after PKCδ inhibition, leading to the activation (phosphorylation) of H2AX, a histone H2A variant. Activation of H2AX was attenuated when JNK1/2 levels were repressed, indicating that H2AX activation is mediated by the JNK pathway in response to PKCδ inhibition. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. To explore the potential of B106 further, melanoma cell lines harboring BRAF mutations that had evolved resistance to a BRAF inhibitor, PLX4032 (vemurafenib), were developed. B106 effectively induced cytotoxicity in these cells, suggesting the potential clinical application of targeting PKCδ in patients who have relapsed following treatment with PLX4032. Taken together, this work suggests that inhibition of PKCδ causes caspase-dependent apoptosis in melanomas with NRAS mutations and in PLX4032-resistant BRAF mutant melanomas. This apoptosis is mediated via activation of the JNK-H2AX pathway, which involves a novel role for phospho-H2AX in the execution of apoptosis.

L'étude des interactions non-covalentes et des relations structure-fonction est à la base de la compréhension des systèmes biologiques. La MS supramoléculaire est une technique de choix pour l'étude des interactions protéine/protéine ou protéine/ligand. Dans le cadre d'études qualitatives ou quantitatives, pour chaque système étudié, les conditions expérimentales et les paramètres instrumentaux ont été optimisés pour conserver le complexe en phase gazeuse (1). L'objectif principal de ce travail est de caractériser le site nucléotidique de hPEBP1 et de contribuer à la découverte de molécules anti-métastases. Sur le plan fonctionnel, une activité enzymatique de hPEBP1 n'a pas pu être mise en évidence. Pour ce projet, une méthode MS de détermination de KD de complexes à faible affinité, plus précise et ne nécessitant par l'utilisation d'un ligand de référence a été développée (2). Une recherche des déterminants structuraux d'un ligand optimal de hPEBP1 a été réalisée par criblage de composés issus d'une synthèse raisonnée basée sur la structure des nucléotides FMN et GTP et par la détermination de leur KD (3). Les criblages ont montré que les critères structuraux indispensables pour la liaison sont la présence d'un groupement chargé ou donneur d'électrons, d'une structure apparentée à une base azotée et d'un cycle additionnel. Une part importante de l'affinité est liée au caractère hydrophobe du ligand. Certains ligands de synthèse ont montré une activité inhibitrice de l'invasion des lignées tumorales.

Leiomiosarcoma maligno e leiomioma benigno rappresentano le due controparti della trasformazione del miometrio. Tuttavia la diagnosi differenziale per discriminare tra lesioni benigne e maligne rappresenta un grande problema. Così vanno identificati nuovi markers per rendere la diagnosi differenziale piu’ accurata. Il ruolo pleiotropico dell’RKIP nel leiomiosarcoma non è ancora chiaro. In questa tesi tramite l’immunoistochimica si sono riscontrate la tendenza di cinque diverse varianti istologiche di lesioni benigne a essere positive all’RKIP e, al contrario, la tendenza delle lesioni maligne a essere negative all’RKIP. Ciò è stato molto evidente confrontando leiomiosarcoma maligno e leiomioma con nuclei bizzarri che, sebbene benigno, ha caratteristiche intermedie tra lesioni benigne e maligne. Per capire se l’RKIP ha un ruolo biologico nel leiomiosarcoma, sono stati eseguiti il knockout in vitro dell’RKIP e l’overespressione in vitro dell’RKIP nelle cellule di leiomiosarcoma. Si è visto che circa la vitalità delle cellule nel tempo e la capacità migratoria, sia le cellule RKIP knockout che le cellule che overesprimono RKIP hanno una tendenza simile a quella dei rispettivi controlli negativi. D'altra parte si è visto che le cellule RKIP knockout hanno una maggiore capacità di formare colonie rispetto al controllo negativo in cui l'espressione dell’RKIP non è stata modificata. In conclusione i risultati ottenuti suggeriscono che può esserci una perdita di RKIP nel leiomiosarcoma maligno e che ciò può favorire clonogenicità. Nel complesso questi risultati suggeriscono che l’RKIP potrebbe candidarsi come marker addizionale per l'analisi differenziale per discriminare se una preparazione simile al leiomioma benigno con nuclei bizzarri non sia, in realtà, una lesione maligna. Alla luce della rarità delle patologie studiate questi risultati costituiscono un punto di partenza per ulteriori ricerche per considerare l’RKIP un marker affidabile a tutti gli effetti.
Malign leiomyosarcoma and benign leiomyoma represent the two counterparts of the myometrium transformation. Nevertheless, the differential diagnosis to discriminate benign from malign lesions represents a remarkable problem. So, it is important to identify new markers making the differential diagnosis more accurate. The pleiotropic RKIP role on leiomyosarcoma is still unclear. In this study, the tendency of five different histological variants of benign lesions to be positive for RKIP and, conversely, the tendency of malign lesions to be negative for RKIP were found by immunohistochemistry. This was particularly evident comparing the malign leiomyosarcoma to the leiomyoma with bizarre nuclei that, although benign, shows intermediate features between benign lesions and malign ones. To understand if RKIP plays a biological role in leiomyosarcoma, in vitro knockout of RKIP and in vitro overexpression of RKIP in leiomyosarcoma cells were performed. It was found that about cells viability overtime and migratory capability both RKIP knockout cells and RKIP overexpressing cells show a trend similar to that of their respective negative controls. On the other hand, it was observed that RKIP knockout cells show a greater ability to form colonies when compared with negative control where the expression of RKIP was not modified. In conclusion, the obtained results suggest that there could be a RKIP loss in the malign leiomyosarcoma and this may favor the clonogenicity. Altogether these results suggest that RKIP may be a candidate to be considered as an additional marker for the differential analysis to discriminate if a preparation similar to the benign leiomyoma with bizarre nuclei, is not, actually a malign lesion. In light of the rarity of the studied pathologies, these results constitute a starting point for further researches in order to consider RKIP a reliable marker in all respects.

Les gains somatiques du chromosome 21 (+21) sont fréquemment observés dans les hémopathies, et les enfants atteints par le Syndrome de Down (DS, trisomie 21 constitutive) ont un risque plus élevé de développer des leucémies durant l’enfance. Ces observations indiquent que +21 participent au développement leucémique. Cependant, la trisomie 21 seule n’est pas suffisante. L'objectif de cette thèse a été de caractériser et modéliser les altérations génétiques coopérant avec les gains du chromosome 21. Un premier axe a permis d’étudier l’impact de la mutation activatrice JAK3A572V dans un modèle murin (knock-in au locus endogène) dans le développement d’un lymphome cutané à cellules T (CTCL). Croisé avec un modèle de trisomie 21 partiel (Ts1Rhr), la latence de ce CTCL est fortement réduite, indiquant une coopération oncogénique. Dans un deuxième axe, j'ai identifié une forte incidence des altérations activant de la voie RAS/MAPK dans les leucémies aiguës lymphoblastiques B pédiatriques (LAL-B) +21. J'ai démontré que la mutation KRASG12D coopère fonctionnellement avec la trisomie 21 dans le processus de transformation à la fois dans des modèles cellulaires murins et des cellules leucémiques humaines. J’ai également développé 20 modèles de xénogreffes pour tester l’efficacité du trametinib, un inhibiteur de la voie RAS/MAPK. Seul ou en combinaison avec des chimiothérapies conventionnelles (vincristine), j’ai montré que l’inhibition de la voie RAS/MAPK prolonge la survie de ces souris. Ces données indiquent que la caractérisation et le ciblage d’événements de coopération permettent de proposer de nouvelles stratégies thérapeutiques pour les leucémies pédiatriques avec +21
Somatic gains of chromosome 21 (+21) are hallmark of hematological malignancies, and children with Down Syndrome (DS, constitutive trisomy 21) are predisposed to develop leukemia. These observations strongly suggest that gains of chromosome 21 promote leukemia development; however, alone, it is not sufficient. The aim of my PhD work was to identify and functionally characterize the genetic alterations cooperating with +21. My first aim was focused on studying the impact of the JAK3A572V activating mutation in the development of cutaneous T cell lymphoma (CTCL), using a new knock-in model carrying this alteration at the endogenous locus. In this study, I showed that partial trisomy 21 (Ts1Rhr) cooperates with the JAK3A572V mutation to reduce the latency of this pathology, thus highlighting a mechanism of oncogenic cooperation. In a second aim, I identified a high incidence of genetic alterations leading to RAS/MAPK pathway activation in B cell leukemia samples carrying +21 (B-ALL+21). I have demonstrated that the KRASG12D mutation functionally cooperates with trisomy 21 in transformation process of both murine and human cellular models. In order to test new molecules to improve the treatment of LAL-B+21, I have also developed 20 xenograft models. Treatment of these models with trametinib, a RAS/MAPK pathway inhibitor, alone or in combination with conventional chemotherapies (vincristine), improve their survival. Together, these data indicate that characterizing and targeting cooperation events allow to propose novel therapeutic strategies in pediatric leukaemia with +21

碩士
國立成功大學
微生物及免疫學研究所
94
Epithelial membrane protein-2 (EMP2), a member of GAS3/PMP22 family, is a tetraspan protein and has been implicated in the control of cell growth, proliferation and cell apoptosis. Dr. N. H. Chow’s study revealed that soy isoflavones could enhance the expression level of EMP2 and inhibited the growth of bladder cancer cells. Besides, in microarray data EMP2 overexpression inhibited Ras related genes, such as Ras homolog gene family, member Q (RHOQ)、member of Ras oncogene family-like 4 (RABL4)、Ras and Rab interactor 3 (RIN3)。Ha-ras mutations are common in bladder carcinomas. It is interesting to reveal whether EMP2 overexpression can inhibit Ras related tumor formation. pEMP2-GFP fusion plasmid was transfected into Ras inducible cell line (7-4) to establish the stable cell lines. The results of the stable cell lines overexpressing EMP2 demonstrated that EMP2 can inhibit Ras activity as well as Ras protein expression and the regulation is at post-transcription level. EMP2 overexpression suppressed Ras induced cell proliferation but not cell migration. Further analysis showed that EMP2 overexpresion can induce cell apoptosis through disruption of the cell cycle progression. EMP overexpression could also suppress Ras induced colony formation. The EMP2 overexpression cells and 7-4 cells were subcutaneously injected into ICR and SCID mice, which are immune competent and immune deficient, respectively. For tumor formation, the tumor size in ICR and SCID mice was 6~27 fold and 3 fold smaller than the control Ras alone induced tumor. These results suggest that EMP2 can inhibit Ras induced tumor formation in mice and host immunity is also involved. Moreover, EMP2 overexpression can inhibit blood vessel formation. All toghther, we reveal that EMP2 overexpression inhibits Ras-related tumorigenesis, possibly through suppressing Ras activity, inducing cell apoptosis and decreasing blood vessels. In conclusion, EMP2 possesses features of tumor suppresser and has the potential to be used to against Ras-related cancers.

Das Raf kinase inhibitor protein (RKIP) ist ein Kinaseregulator, der im Herzen eine Präferenz für die G-Protein-gekoppelte Rezeptorkinase 2 (GRK2) zeigt. Die Regulation erfolgt durch direkte Interaktion beider Proteine, wird durch eine PKC-Phosphorylierung an Serin 153 des RKIP induziert und inhibiert die GRK2-vermittelte Phosphorylierung von G-Protein-gekoppelten Rezeptoren (GPCR). Die GRK2 desensitiviert GPCR und eine Hemmung der GRK2-Aktivität wirkt sich so positiv auf die Ansprechbarkeit von GPCR aus. Die \textbeta-adrenergen Rezeptoren (\textbeta AR) sind im Herzen maßgeblich an der Regulation der kardialen Kontraktilität beteiligt. Erste Zusammenhänge zwischen der RKIP-Expression und der kontraktilen Antwort von Kardiomyozyten wurden bereits in einer früheren Arbeit untersucht und bestätigt. Sie begründen die Fragestellung nach Effekten einer verstärkten RKIP-Expression auf \textbeta-adrenerge Rezeptorsignale, Herzfunktion und die Entwicklung der Herzinsuffizienz. Im Rahmen dieses Projektes konnten die Effekte des RKIP auf \textbeta-adrenerge Signalwege detaillierter beschrieben werden. Dabei erwies sich die inhibitorische Funktion auf die GRK2 als rezeptorspezifisch ohne Einfluss auf zytosolische Angriffspunkte der GRK2 zu nehmen. Verstärkte \textbeta-adrenerge Signale zeigten sich in neonatalen Kardiomyozyten an Hand der erhöhten cAMP-Level, PKA-Aktivität, sowie Kontraktionsrate und Relaxationsgeschwindigkeit nach \textbeta-adrenerger Stimulation. Im Einklang damit konnte eine erhöhte PKA- und CaMKII-Aktivität und eine positive Inotropie in transgenen Tieren, mit herzspezifischer Überexpression von RKIP, beobachtet werden. Durch Messung des Calcium-\textit{Cyclings} in Kardiomyozyten konnte der Phänotyp auf eine verbesserte Rückführung des Calciums, einer daraus resultierenden erhöhten Calciumbeladung des sarkoplasmatischen Retikulums und einem gesteigerten systolischen Calciumspiegel, zurückgeführt werden. Die Untersuchung der Phosphorylierung von Calciumkanälen, L-Typ-Calciumkanal und Ryanodin-Rezeptor 2, die den einwärtsgerichteten Calciumstrom vermitteln konnte ihre Beteiligung an der positiv inotropen Wirkung ausschließen. Neben dem kontraktilen Phänotyp konnten zusätzliche protektive Effekte beobachtet werden. In Modellen, die eine chronische \textbeta-adrenerge Stimulation imitieren, bzw. eine Nachlasterhöhung induzieren konnte eine Verringerung der interstitiellen Fibrose und der damit assoziierten Marker, gezeigt werden. Mit Hilfe von \textit{in vivo} EKG-Messungen konnte die Neigung zur Ausbildung von Arrhythmien untersucht werden. Auch im Hinblick auf die Anzahl der Extrasystolen waren RKIP-transgene Tiere geschützt. Infolge der Untersuchung der Phänotypen in Deletionshintergründen der einzelnen \textbeta AR-Subtypen (\textbeta\textsubscript{1}AR, \textbeta\textsubscript{2}AR) konnte die positive Inotropie mit den spezifischen Signalwegen des \textbeta\textsubscript{1}AR assoziiert und die protektiven Effekte gegenüber den Umbauprozessen und der Arrhythmieneigung dem \textbeta\textsubscript{2}-adrenergen Signalen zugeschrieben werden. Zusätzlich bestätigt sich eine besondere Rolle der G\textalpha\textsubscript{i}-Kopplung des \textbeta\textsubscript{2}AR, durch die er einen hemmenden Einfluss auf die \textbeta\textsubscript{1}AR-Singale nehmen kann. Die Untersuchung einiger Marker, die eine physiologische von einer pathologischen Hypertrophie unterscheiden, konnte das in den RKIP-transgenen Mäusen auftretende Wachstum der Kardiomyozyten als kompensatorische und physiologische Hypertrophie charakterisieren. Zusammengenommen weisen diese Ergebnisse auf eine ausgeglichene Aktivierung der beiden Rezeptoren hin, die sich gegenseitig regulieren und durch die Inhibition der GRK2 in ihrer Anregbarkeit erhalten bleiben. Mittels einer AAV9-vermittelten Gentherapie konnte das therapeutische Potential dieses Prinzips weiter bestätigt werden, da es die prominentesten Veränderungen während der Herzinsuffizienzentwicklung, wie die Verschlechterung der linksventrikulären Funktion, die Dilatation des linken Ventrikels, die Ausbildung von Lungenödemen und interstitieller Fibrose sowie die Expression von Herzinsuffizienz-assoziierten Genen, verhindern konnte. Auch konnten die Auswirkungen der Deletion des RKIP, die sich durch eine beschleunigte und gravierendere Herzinsuffizienzentwicklung auszeichnet, durch Reexpression von RKIP verhindert werden. Diese Arbeit kann somit zeigen, dass das RKIP eine ausgeglichene Verstärkung von \textbeta-adrenergen Signalwegen verursacht, die positiv inotrop und gleichzeitig protektiv wirkt. Dieses Wirkprinzip könnte ferner eine Strategie zur Erhöhung der Kontraktilität in der Herzinsuffizienz darstellen, die entgegen etablierter Theorien auf der Stimulation beider \textbeta AR basiert
The Raf kinase inhibitor protein (RKIP) is a kinase regulator with a preference for the G protein-coupled receptor kinase 2 (GRK2) in the heart. The mechanism is a direct interaction of GRK2 and RKIP, which is triggered by a PKC-mediated phosphorylation at serine 153 of RKIP. By binding the GRK2, RKIP prevents the GRK2-mediated GPCR-phosphorylation and, thus, desensitisation of GPCR. As a result, inhibition of GRK2-activity positively affects the responsiveness of cardiac G protein-coupled receptors (GPCR). The GPCR primarly responsible for the regulation of the cardiac contractility are the \textbeta-adrenergic receptors (\textbeta AR). Previous work proved an interrelation of RKIP-expression and contractile response of cardiomyocytes and set a basis for the subject of this thesis, dealing with the effects of RKIP-expression on beta-adrenergic signalling, cardiac function and the development of heart failure. The work describes the impact of RKIP on \textbeta-adrenergic signaling in more detail. An important feature of the inhibitory function of RKIP on GRK2 is a specificity for receptor targets (\textbeta AR) with no, or only minor, impact on the cytosolic targets of the GRK2. RKIP also increases \textbeta-adrenergic signalling. This appears in neonatal cardiac myocytes through an increased cAMP-generation, PKA-activity, contractile action and relaxation velocity after \textbeta-adrenergic stimulation. Similarly, RKIP-transgenic mice, with heart specific RKIP-expression, showed higher PKA and CaMKII-activities as well as, a positive inotropy. Analysis of the calcium cycling in these cardiomyocytes provided an explanation for the hypercontractile phenotype: an enhanced calcium reuptake into the sarcoplasmatic reticulum (SR), the resulting higher calcium load of the SR and an increased calcium amplitude in the cytosol during the systole cause the augmented contractile force. Furthermore, it could be ruled out, that two inward rectifying channels - L-type calcium channnel and Ryanodin Receptor 2 contribute to the positve inotropy in RKIP-transgenic mice. Besides, the RKIP-expression had additional protective effects in heart failure development, which were investigated by desease models. Hypertrophy was induced by chronic \textbeta-adrenergic stimulation and heart failure by induction of pressure overload. Under these conditions, RKIP could reduce the development of interstitial fibrosis and the expression of associated marker genes. The occurence of arrhythmias, in particular ectopic beats, was assessed by the analysis of \textit{in vivo} ECG-traces. Rated by the number of ectopic beats RKIP-transgenic mice were also protected against the induction of arrhythmia. The analysis of RKIP-expression in \textbeta AR subtype-KOs (\textbeta\textsubscript{1}KO, \textbeta\textsubscript{2}KO) could relate the different effects of RKIP to the signalling pathways of either \textbeta\textsubscript{1}AR or \textbeta\textsubscript{2}AR. As a result, RKIP effects the positive inotropy through signals of the \textbeta\textsubscript{1}AR and the protection against heart failure-related remodelling processes and arrhythmia through signals of the \textbeta\textsubscript{2}AR. Additionally a major importance could be assigned to the G\textalpha\textsubscript{i} coupling of the \textbeta\textsubscript{2}AR. This capacity of the \textbeta\textsubscript{2}AR can counteract potentially maladaptive signalling of the \textbeta\textsubscript{1}AR. A monitored growth of cardiomyocytes of RKIP-transgenic mice was assessed in greater depth using different markers to differentiate physiological from pathological hypertrophy. Thereby the occurring hypertrophy was characterised as physiological and compensatory. Taken together, these results point towards a balanced activation of both \textbeta AR. They influence each other through downstream signals and are protected from desensitisation and loss of \textbeta-adrenergic responsivness through inhibition of the GRK2 by RKIP. To validate the therapeutic potential of this mode of action, an AAV9-mediated gene therapy was conducted. In this setting, RKIP was able to prevent, or strongly reduce the most prominent changes during heart failure development. Among these are the decline of the left ventricular function, dilation of the left ventricle, development of a pulmonary congestion, interstitial fibrosis and the expression of heart failure associated genes. Moreover, the consequences of RKIP deletion, which are reflected in an accelerated and deteriorated heart failure development, could be reversed by the reexpression of RKIP. This work shows, that RKIP induces an even activation of \textbeta-adrenergic signalling, which results in a positive inotropy with concomitant protective effects. RKIPs mode of action represents a strategy and bears the possibilty to enhance cardiac contractility in the failing heart by stimulation of both \textbeta AR, which is contrary to the common belief

RKIP reguliert Proteinkinasen der Signaltransduktionskaskaden von G Protein-gekoppelten Rezeptoren, der Raf/MEK/ERK-MAPK, des Transkriptionsfaktors NFκB und von GSK3β. Unklar war bisher, wie die spezifische Interaktion von RKIP mit seinen mannigfaltigen Interaktionspartnern ermöglicht und reguliert wird. Raf1 und GRK2 sind die einzigen bekannten direkten Interaktionspartner von RKIP und wurden deshalb gewählt, um die zugrundeliegenden molekularen Mechanismen dieser Interaktion genauer zu untersuchen. In dieser Arbeit wurde gezeigt, dass RKIP nach PKC-vermittelter Phosphorylierung von Serin153 dimerisiert und dass diese Dimerisierung für die RKIP/Raf1-Dissoziation und die RKIP/GRK2-Interaktion essentiell ist. Co-Immunpräzipitationsexperimente mit einer phosphorylierungsdefizienten Mutante zeigten, dass für diese Dimerisierung die Phosphorylierung von beiden RKIP-Molekülen notwendig ist. Als Dimerinteraktionsfläche wurden die Aminosäuren 127-146 von RKIP identifiziert, da das Peptid RKIP127-146 die Dimerisierung von RKIP spezifisch und effizient hemmte. Um die Bedeutung dieser phosphorylierungsinduzierten Dimerisierung von RKIP für seine Interaktion mit Raf1 und GRK2 zu untersuchen, wurden eine phosphomimetische Mutante (RKIPSK153/7EE) und eine Mutante von RKIP generiert, welche bereits unphosphoryliert dimerisiert (RKIP∆143-6). Folgende Ergebnisse legen nahe, dass die Dimerisierung von RKIP für die spezifische Interaktion mit Raf1 bzw. GRK2 entscheidend ist: (i) Die Dimerisierung von phosphoryliertem RKIP ging mit der Dissoziation von RKIP und Raf1 und der Assoziation von RKIP und GRK2 einher; (ii) die Mutanten RKIPSK153/7EE und RKIP∆143-6, die bereits in unstimulierten Zellen eine starke Dimerisierung zeigten, hatten eine höhere Affinität zu GRK2 als zu Raf1; (iii) die Hemmung der RKIP-Dimerisierung interferierte nur mit der RKIP/GKR2- aber nicht mit der RKIP/Raf1-Interaktion; (iv) in vitro und in Mausherzen konnte ein RKIP- und GRK2-immunreaktiver Komplex nachgewiesen werden; (v) Untersuchungen zur RKIP-vermittelten Hemmung der Kinaseaktivität von GRK2 und Raf implizierten, dass dimerisiertes RKIP nur die Aktivität von GRK2, nicht aber von Raf hemmt. Diese Arbeit zeigt, dass die phosphorylierungsinduzierte Dimerisierung von RKIP die spezifische Interaktion von RKIP mit Raf1 und GRK2 koordiniert. Die Aufklärung dieses Mechanismus erweitert unser Verständnis der spezifischen Interaktion von Kinasen mit ihren Regulatorproteinen
RKIP is a regulator of several distinct kinases and modulates diverse signal transduction cascades such as the signaling of G protein coupled receptors, of the Raf/MEK/ERK-cascade, of the transcription factor NFκB, and of GSK3β. Until now, it was not well understood how the specific interaction of RKIP with its diverse targets is achieved and regulated. Raf1 and GRK2 are the only known direct interaction partners of RKIP and were thus chosen to untangle the molecular mechanisms regulating the specific interaction of RKIP with these kinases. In this dissertation it is shown that RKIP dimerizes upon PKC-mediated phosphorylation of serine153 and that this dimerization is essential for RKIP/Raf1-dissociation and RKIP/GRK2-association. Co-immunoprecipitation experiments with a phosphorylation-deficient mutant revealed that the dimerization of RKIP requires the phosphorylation of two RKIP molecules. The amino acids 127-146 of RKIP were identified as dimer-interface, since RKIP-dimerization was efficiently and specifically inhibited by the peptide RKIP127-146. To elucidate the implication of this phosphorylation-induced dimerization on the target specificity of RKIP, a phosphomimetic RKIP mutant (RKIPSK153/7EE) and a dimeric RKIP mutant (RKIP∆143-6) were generated. The following results indicated that dimerization of RKIP controls its specific interaction with Raf1 or GRK2, respectively: (i) dimerization of phosphorylated RKIP occurred concomitantly with the release of RKIP from Raf1 and its association with GRK2; (ii) the RKIP mutants RKIPSK153/7EE and RKIP∆143-6, which had a higher propensity for RKIP-dimerization already under basal conditions, had a higher affinity to GRK2 than to Raf1; (iii) inhibition of RKIP-dimerization prevented only RKIP/GRK2-binding but did not interfere with RKIP/Raf1-binding; (iv) an RKIP- and GRK2-immunoreactive complex was detected in vitro as well as in mouse hearts; (v) analyses of RKIP-mediated inhibition of GRK2 and Raf showed that a higher propensity for RKIP-dimerization translates into efficient GRK2-inhibition but not into Raf-inhibition. The results of this thesis show that phosphorylation-induced dimerization of RKIP regulates its specific interaction with Raf1 and GRK2. The elucidation of this mechanism improves our understanding how specificity in the interaction of kinases and their regulatory proteins can be achieved