Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19243号 / 農博第2140号 / 新制||農||1036(附属図書館) / 学位論文||H27||N4947(農学部図書室) / 32242 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 祝前 博明, 教授 松井 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

專利制度是知識經濟時代最為重要的一種智慧財產權形式，不但對於技術創新居功厥偉，在國際商業活動中也占有極具份量的地位。而在取得專利的三大要件中，以非顯而易知性（即我國進步性）最為棘手，蓋其本身屬於不確定之法律概念，而容有裁量空間。 非顯而易知性發軔於美國判例法，其後由實務主導其發展。在指標性案例KSR判決中，最高法院揭示了非顯而易知性的審查架構，以Graham四要件法則為根柢，並輔以顯可嘗試原則及彈性運用的TSM檢測法，整體而言KSR判決提高了非顯而易知性的適格門檻。在後KSR時代，CAFC在機械工業、醫藥品與生物科技等領域分別依不同程度適用KSR見解。2009年In re Kubin案確認KSR見解可適用於不可預測性較高之基因生技領域，近幾年來顯可嘗試原則也獲得高度重視。 相較而言，我國進步性審查主要依據智慧財產局所制定的專利審查基準，但行政審查常有過於直觀簡略之嫌；法院判決則在「發明所屬領域中具通常技術者之技術水準」與「該領域具通常技術者參酌先前技術所揭露之內容及申請時的通常知識，是否能所能輕易完成系爭申請發明之整體」此兩步驟的論證上較為欠缺，整體而言達成進步性結論之心證揭露程度不足，對於當事人有突襲性裁判之虞。 本研究基於上述觀察所得，對美國與我國關於非顯而易知性概念之認知與實踐進行比對，並分別就審查實務面與產業因應面提出微薄建議，以期借鏡美國法經驗使我國未來實務操作更趨完善。 / Patent system is one of the most important forms of intellectual property rights in the era of knowledge economy, not only indispensable for technological innovation, also of great influnce in the international business activities. Among the three requirements of patentability, “Non-obviousness” (ie, “Inventive Step” in Taiwan) is the most difficult to fulfill, due to the uncertainty of its legal concept and the room for discretion. Non-obviousness was carved out in the U.S. case law and continuously developed by the court rulings. In the benchmark case KSR v. Teleflex, the Supreme Court articulated that the examination framework of non-obviousness is based on Graham four factors, along with other principles like “Obvious to Try” and the TSM test in a more flexible way. In general, KSR lifted the eligibility threshold for non-obviousness. It has been applied in different degrees by the Court of Appeals for the Federal Circuit to various fields such as machinery industry, pharmaceuticals and biotechnology in the post-KSR era. Then it was recognized in 2009 In re Kubin case that the KSR opinion is applicable to the unpredictable field, gene biotechnology, for instance. Besides, the “Obvious to Try” principle has been gaining much attention in recent years. In comparison, both administrative and juducial examinations of inventive step in Taiwan are mainly based on the “Substantive examination guidelines for invention patent” issued by the Intellectual Property Office. However, the administrative review is often reckoned to be too intuitive and rough, and the court decisions are considered to be made with less expression on “the level of the PHOSITA” and “whether a PHOSITA with the reference to prior arts and common knowledge can complete the whole invention without difficulty.” In all, the lack of revealing the reasoning on the inventive step conclusion might expose the parties in danger of surprise judgements. Based on the above observations, this study compared the cognition and practice of non-obviousness both in the United States and in Taiwan, and as a result, presented some primary suggestions in light of the United States’ experience toward both the practice and industries, so that our inventive step examination practice in the future could be improved.

The RAS/RAF/MEK/ERK pathway is the prototypical cellular signal transduction cascade and has been the focus of intense scrutiny over the last two decades. As a mitogenic pathway, its activation is a potent driver of cellular growth and survival, and its deregulation underlies many cancers. While RAS family GTPases have long been recognized as prolific human oncogenes, a landmark study in 2002 also established the RAF family kinase as a bona fide oncogene (Davies et al., 2002). Indeed, aberrant RAS-RAF signaling underlies nearly one-third of all human cancers (Wellbrock et al., 2004). Notably, mutations in RAF are found with astounding frequency in certain cancers (e.g. 70% of malignant melanomas) (Dhomen and Marais, 2007). These findings have identified intercepting aberrant RAF function as an ideal therapeutic target. RAF is a Ser/Thr protein kinase and its activity is strictly regulated by a core complex of at least three proteins, namely, KSR, CNK and HYP (Claperon and Therrien, 2007). The mechanism by which the KSR/CNK/HYP complex regulates RAF function remains enigmatic. In particular, the function of KSR in regulating RAF activity is highly controversial. The work described in this thesis was conducted with the aim of: i) understanding the interactions that underlie formation of the KSR/CNK/HYP complex, and ii) elucidating the mechanism by which the complex regulates RAF function. I have attempted to accomplish these aims using a combination of structural biology, biochemistry and cell biology approaches. I begin by presenting the structure of the SAM domain mediated interaction between CNK and HYP. I describe a model for how the CNK/HYP interaction in turn serves to recruit KSR to form the higher-order KSR/CNK/HYP complex. Subsequently, I describe the allosteric mechanism by which KSR controls RAF activation via the formation of specific side-to-side kinase domain heterodimers of KSR and RAF. Lastly, I describe a potential mechanism by which RAS directly mediates the attainment of the side-to-side dimer configuration of RAF through its own ability to form dimers. The acquisition of the side-to-side dimer configuration is essential for aberrant RAF signaling in cancers, suggesting future RAF inhibition strategies could be aimed at preventing dimer formation.

The RAS/RAF/MEK/ERK pathway is the prototypical cellular signal transduction cascade and has been the focus of intense scrutiny over the last two decades. As a mitogenic pathway, its activation is a potent driver of cellular growth and survival, and its deregulation underlies many cancers. While RAS family GTPases have long been recognized as prolific human oncogenes, a landmark study in 2002 also established the RAF family kinase as a bona fide oncogene (Davies et al., 2002). Indeed, aberrant RAS-RAF signaling underlies nearly one-third of all human cancers (Wellbrock et al., 2004). Notably, mutations in RAF are found with astounding frequency in certain cancers (e.g. 70% of malignant melanomas) (Dhomen and Marais, 2007). These findings have identified intercepting aberrant RAF function as an ideal therapeutic target. RAF is a Ser/Thr protein kinase and its activity is strictly regulated by a core complex of at least three proteins, namely, KSR, CNK and HYP (Claperon and Therrien, 2007). The mechanism by which the KSR/CNK/HYP complex regulates RAF function remains enigmatic. In particular, the function of KSR in regulating RAF activity is highly controversial. The work described in this thesis was conducted with the aim of: i) understanding the interactions that underlie formation of the KSR/CNK/HYP complex, and ii) elucidating the mechanism by which the complex regulates RAF function. I have attempted to accomplish these aims using a combination of structural biology, biochemistry and cell biology approaches. I begin by presenting the structure of the SAM domain mediated interaction between CNK and HYP. I describe a model for how the CNK/HYP interaction in turn serves to recruit KSR to form the higher-order KSR/CNK/HYP complex. Subsequently, I describe the allosteric mechanism by which KSR controls RAF activation via the formation of specific side-to-side kinase domain heterodimers of KSR and RAF. Lastly, I describe a potential mechanism by which RAS directly mediates the attainment of the side-to-side dimer configuration of RAF through its own ability to form dimers. The acquisition of the side-to-side dimer configuration is essential for aberrant RAF signaling in cancers, suggesting future RAF inhibition strategies could be aimed at preventing dimer formation.

La voie de signalisation RAS-ERK régule la prolifération et la différenciation cellulaire par la propagation séquentielle d’un signal jusqu’au noyau, aboutissant à la régulation des gènes cibles. Après réception d’un stimulus extracellulaire conduisant à l’activation de la petite GTPase RAS (Rat Sarcoma), la transduction du signal s’effectue par les phosphorylations successives de RAF (Rapid Accelerated Fibrosarcoma), MEK (MAPK/ERK Kinase 1/2) et ERK (Extracellular signal-Regulated Kinase 1/2). Chez les mammifères, la famille élargie des protéines RAF comprend les trois kinases ARAF, BRAF, CRAF et les pseudokinases KSR1, KSR2 pour Kinase Suppressor of Ras 1/2. En l’absence de stimuli, les kinases RAF adoptent une forme auto-inhibée où leur région régulatrice N-terminale (N-terminal Region ou NTR) inhibe l’activité catalytique de leur domaine kinase (Kinase Domain ou KD). L’activation des GTPases RAS ancre les kinases RAF à la membrane plasmique via le domaine RBD (Ras Binding Domain) de leur NTR. Ce phénomène favorise la dérépression des KD et dévoile leur interface de dimérisation. L’association de deux protéines RAF l’une avec l’autre induit l’activation des kinases RAF et la phosphorylation de leur substrat MEK. Bien que dénuées d’activité kinase intrinsèque, les pseudokinases KSR sont néanmoins capables de dimériser avec les kinases RAF et de les activer. Des mutations des protéines clés de la voie RAS-ERK conduisent à son activation anormale et sont directement responsables du développement et de la progression tumorale. Notamment, la kinase BRAF est altérée dans 7 % des cancers. L’échec des stratégies thérapeutiques permettant d’inhiber les kinases RAF a mis en lumière l’importance de la dimérisation dans la régulation de leur activité. Ainsi, les processus favorisant la formation d’hétérodimères RAF/KSR ne sont, à ce jour, pas bien compris. La problématique de la thèse a été d’identifier les mécanismes moléculaires régissant la formation spécifique du dimère BRAF/KSR1 aboutissant à la phosphorylation du substrat MEK1. Les objectifs de la thèse ont donc été 1) de déterminer ce qui permet au substrat MEK1 de se lier aux différentes protéines de la famille RAF, 2) d’identifier les domaines nécessaires à l’interaction spécifique de BRAF et KSR1, 3) de développer des stratégies de purification du dimère BRAF/KSR1 pour en faire une analyse structurale. Ce travail a dans un premier temps montré que le substrat MEK1 est l’activateur de sa propre kinase BRAF, en favorisant sa transactivation par KSR1 via des interactions au niveau des domaines kinases. De manière inattendue, nous avons par la suite établi que ce sont les NTR des protéines BRAF et KSR1 qui guident leur hétérodimérisation. Le dimère BRAF/KSR1 repose ainsi sur l’interaction directe du domaine BRS de BRAF et du domaine CC-SAM de KSR1. Nous avons montré que le domaine CRD de BRAF exerce une influence sur l’interaction BRS/CC-SAM et par extension, sur la dimerisation de BRAF avec KSR1. Enfin, nous avons testé plusieurs stratégies de purification du dimère BRAF/KSR1 qui nous ont permis d’optimiser une technique de purification à partir de cellules de mammifères et de générer des constructions pour des cellules d’insectes. Ainsi, ce travail nous a permis d’améliorer la compréhension des mécanismes de formation de l’hétérodimère BRAF/KSR1 et son lien avec le substrat MEK1. Nous avons découvert des nouveaux moyens de régulation de la signalisation RAS-ERK. À terme, les résultats obtenus s’avèreront utiles pour le développement de nouvelles stratégies thérapeutiques efficaces pour inhiber la voie RAS-ERK dans des contextes pathologiques. / The RAS-ERK signaling pathway regulates cell proliferation and differentiation by signal propagation from the cell surface to the nucleus, resulting in the regulation of targeted genes. After receiving an extracellular stimulus leading to the activation of the small GTPase RAS (Rat Sarcoma), signal transduction is mediated by the successive phosphorylations of RAF (Rapid Accelerated Fibrosarcoma), MEK (MAPK/ERK Kinase 1/2) and ERK (Extracellular signal-Regulated Kinase 1/2) kinases. In mammals, the extended family of RAF proteins is comprised of the three kinases ARAF, BRAF, CRAF and the two pseudokinases KSR1, KSR2 from Kinase Suppressor of Ras 1/2. In the absence of a stimulus, RAF kinases are in an auto-inhibited conformation wherein their N-terminal regulatory region (NTR) inhibits the catalytic activity of their kinase domain (KD). Activation of the RAS GTPases anchors RAF kinases to the plasma membrane through binding of the RBD (Ras Binding Domain), present in their NTR. This phenomenon induces the release of the KDs and unveils their dimerization interfaces. The association of two RAF proteins with each other stimulates the activation of RAF kinases and the phosphorylation of their substrate MEK. Although lacking an intrinsic kinase activity, KSR pseudokinases are nevertheless able to stimulate RAF kinase activity through dimerization and transactivation. Mutations of core members of the RAS-ERK pathway led to its abnormal activation and are directly responsible for tumor development and progression. In particular, the BRAF isoform is mutated in 7 % of cancers. Unsuccessful therapeutic strategies developed to inhibit RAF kinases have highlighted the importance of dimerization in the regulation of the catalytic activity of RAF kinases. Moreover, the process favoring the formation of RAF/KSR heterodimers is not fully understood. The focus of this Ph.D. was to identify the molecular mechanisms governing the specific formation of the BRAF/KSR1 dimer leading to the phosphorylation of the MEK1 substrate. Our main objectives were therefore to 1) determine what allows the substrate MEK1 to bind to the different members of the RAF family of proteins, 2) identify the domains necessary for the specific interaction of BRAF and KSR1 3) develop a new approach to purify the BRAF/KSR1 dimer for structural analysis. This work showed that the substrate MEK1 stimulates the activation of its own kinase, by promoting BRAF transactivation by KSR1 through interactions at the kinase domain level. Unexpectedly, we subsequently established that the NTRs of BRAF and KSR1 guide their heterodimerization. BRAF/KSR1 dimer formation is thus based on direct interaction of the BRS domain of BRAF and the CC-SAM domain of KSR1. We then showed that the CRD domain of BRAF has an influence on the BRS/CC-SAM interaction which overall modulates the dimerization of BRAF with KSR1. Finally, we tested several BRAF/KSR1 dimer purification strategies that allowed us to optimize a purification technique from mammalian cells. We also generated constructs enhanced for insect cells expression in the hope of successfully stabilizing BRAF/KSR1 in a signaling complex. Thus, this work allowed us to improve the understanding of the mechanisms underlying the formation of BRAF/KSR1 heterodimer and its link with its MEK1 substrate. We have discovered new ways of regulating the RAS-ERK signaling pathway. Ultimately, theses results will prove useful for the development of new effective therapeutic strategies to inhibit the RAS-ERK pathway in pathological contexts.

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