Glioblastoma (GBM) is a very aggressive and invasive tumour that relapses within nine months of diagnosis and remains incurable despite advances in multimodal therapy including surgical resection, chemotherapy and radiation. Poor patient outcome has been correlated to specific markers of brain tumour initiating cells (BTIC) and intratumoural heterogeneity (ITH), which have also been associated with treatment resistance and tumour recurrence. ITH can be explained at the cellular level by the existence of multiple populations of cancer cells, including some which have acquired stemness properties like self-renewal, proliferation, and multilineage differentiation, also known as cancer stem cells (CSCs). In brain tumours, CSCs or BTICs, have been shown to be resistant to both chemotherapy and radiation treatment, allowing them to escape therapy and consequently generate for tumour recurrence. As a result, therapies that focus on targeting the BTIC compartment within the bulk GBM tumour would provide better treatment and prognosis for patients. To profile GBM BTICs we conducted two transcriptomic screens. The first compared GBM BTICs to neural stem cells (NSCs), their healthy counterparts, and for the second we developed a pipeline utilizing a dynamic BTIC patient-derived xenograft (PDX) model of human GBM recurrence allowing for the profiling of GBM BTICs at engraftment, after chemoradiotherapy delivery in a phase we have termed "minimal residual disease" (MRD), and at tumour recurrence. In this study, Dickkopf-1 (DKK1) was identified as a potential therapeutic target for GBM from each transcriptomic screen and was studied using short hairpin knockdowns, blockade with monoclonal antibodies, and subsequent functional stem cell assays. / Thesis / Master of Science (MSc) / Glioblastoma (GBM) is a very aggressive tumour that relapses within nine months of diagnosis and remains incurable despite chemotherapy, radiation, and surgery. Relapse is believed to be caused by the presence of a wide variety of cell types, including cancer stem cells (CSCs), which have been shown to be resistant to both chemotherapy and radiation in GBM. As a result, therapies that focus on targeting the CSCs within the bulk GBM tumour would provide better treatment for patients. In this study, we analyzed this cell population by conducting two screens. The first compared the level at which genes are expressed in GBM CSCs in comparison to how they are expressed in their healthy counterparts, neural stem cells, whereas the second compared the primary patient GBM tumour to its relapsed form in a mouse model of the disease. In this study, the protein Dickkopf-1 (DKK1) was identified and validated as a potential therapeutic target of GBM using well established molecular and stem cell functional assays.

Hematopoiesis is the highly regenerative process of producing billions of blood cells each day, including white blood cells, red blood cells, and platelets. Given the relatively short life span of these mature cells, hematopoiesis is dependent on stem and progenitor cells to generate renewed progeny, which represents a tightly regulated process. This includes cell intrinsic and external factors, and where dysregulation can lead to anemia and cancer. As such, the hematopoietic hierarchy has been intensely studied for nearly a century and represents a gold standard model of cell fate and developmental biology, in research and clinical applications. Cellular models, such as in vitro culture and human-mouse xenografts in vivo, have been developed to explain complex phenomena pertaining to hematopoiesis and also interrogate processes which are too invasive to study in humans. Hematopoietic generation is required beyond sustaining homeostasis, and progenitors can be damaged through cytotoxic injuries such as radiation and standard chemotherapy, and also undergo leukemic transformation. There are two main treatment modalities for leukemia patients (a) receiving a stem cell transplant, and (b) drug or radiation-based therapy. In the former, shortages of donors and stem cells has remained an unmet clinical need for decades. In the latter, selective targeting of genetic mutations has become a successful standard-of-care in leukemias such as chronic myelogenous leukemia and acute promyelocytic leukemia. However, in the most common adult hematologic malignancy, chronic lymphocytic leukemia (CLL), similar targeting therapies have not been developed. Altogether, shortages of stem cells from healthy donors, chemotherapy-induced immune dysfunction, and a lack of targeted therapies, all reinforce the immediate need for innovative cellular models to address these clinical problems. To generate additional sources of human hematopoietic progenitors for laboratory study, human PSCs have been used. Unlike hematopoietic progenitor cells collected from healthy and leukemic donors, human pluripotent stem cells (PSC) can be easily propagated and expanded in vitro. PSCs can generate hematopoietic progenitor cells, but they remain poorly understood and have not been robustly applied to solve the aforementioned deficiencies related to patient treatment. Importantly, the biological regulation of both hematopoiesis and PSCs has been experimentally confirmed to significantly deviate between humans and other animals, such as mice, further reinforcing the importance of human-specific cell models of hematopoiesis. Therefore, I hypothesized that human stem cell models provide a focused approach to interrogate the regulation of hematopoiesis from the apex of the hierarchy, which can be used to understand the promotion of healthy hematopoiesis and understand malignant transformation. Collectively, the data presented within this thesis offer a deeper conceptualization of human stem cell models and the deconvolution of several complex components of hematopoietic regulation. This work has revealed novel, clinically relevant, and actionable targets to ultimately enable the promotion of healthy hematopoiesis on multiple fronts. / Thesis / Doctor of Philosophy (PhD) / This thesis presents research on novel molecular and genetic regulatory pathways of self-renewal and differentiation in models of healthy and malignant human hematopoiesis. The origin of healthy hematopoietic regulation stems from a large body of work spanning decades and encompasses many efforts by others to derive hematopoietic stem cells from human pluripotent cells. The development of a genetic model for the malignant regulation of CLL was truly serendipitous, was propelled through robust and intriguing results that begged for further exploration, and filled a clinical gap in identifying actionable targets in CLL. Lastly, these two projects, along with my supportive roles in other published works throughout my graduate studies, instructed me to develop a human-mouse transplant model to uncover the biology of regenerating healthy hematopoiesis during injury.

Final publication is available at http://www.bloodjournal.org/ / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19614号 / 医博第4121号 / 新制||医||1015(附属図書館) / 32650 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 山田 亮, 教授 三森 経世 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Neuartige Vakzinierungsstrategien zur Aktivierung einer Tumor-spezifischen zellulären Immunantwort sind vielversprechende Ansätze zur Therapie von Tumoren, insbesondere Human Papillomvirus (HPV)-assoziierte Tumore. Bisherige HPV-Impfstudien zeigen zwar die Aktivierung einer spezifischen zellulären Immunantwort, eine Tumorreduktion bleibt jedoch aus. Um diesen Effekt auf Immunzellebene zu definieren, wurde die Wirkung der HPV-Vakzine Ad p14 im Mausmodell und an Untersuchungsmaterial humaner Tumore analysiert. In Mäusen bildeten sich HPV+ TC1-Tumore einer frühen Entwicklungsphase nach Vakzinierung zurück. Tumore einer späten Entwicklungsphase wuchsen dagegen in zwei Intervallen aus. Immunologische Eigenschaften der Tumorzellen blieben dabei unverändert. Unterschiede zeigten sich in den Frequenzen Tumor-infiltrierender Lymphozyten; in progressiven Phasen wurden nur CD4+ T Zellen nachgewiesen, in Regressionsphasen zusätzlich zytotoxische CD8+ T Zellen. Immunmodulatoren, wie Interferon alpha oder DTA-1, einem Antikörper für den Glucocorticoid-induzierten Tumornekrosefaktor-Rezeptor, unterstützten die Wirkung der Vakzine; letzterer erhöhte die Anzahl zytotoxischer CD8+ T Zellen und führte zur Abstoßung der TC1-Tumore. HPV+ Tumorgewebe des Menschen, wie auch ihre Vorstufen, zeigten im Vergleich zu anderen Tumoren, wie Bronchial oder Kolonkarzinomen einen signifikant höheren Anteil an CD4+ und CD8+ T Zellen und an Forkhead Box P3+ regulatorischen T Zellen. Die Ergebnisse deuten darauf hin, dass die immunologischen Abläufe bei der Entwicklung HPV-assoziierter Tumore mit denen vorangeschrittener chronischer Erkrankungen vergleichbar sind, in denen sich CD4+ und CD8+ T Zellantworten erschöpfen während sich gleichzeitig immunsuppressive Mechanismen verstärken. Um die Entwicklung von Impfstoffen zur Therapie HPV-assoziierter Tumore zu verbessern sollten diese Mechanismen ausführlicher betrachtet werden. / Novel vaccination strategies, activating cellular tumour specific immune responses represent a promising approach for the treatment of cancer. Especially featured for these treatments are tumours evolving from chronic human papillomavirus (HPV) infections. But current strategies have not yet proved efficacious for complete tumour regression. Addressing cellular immunological aspects of tumour vaccination, this work focused on effects of HPV vaccine Ad p14 in mice and in samples of human tumours. In mice vaccination resulted in complete regression of early stage murine HPV+ TC1 tumours. Late stage TC1 tumours increased discontinuously. During that process, TC1 cells preserved their immunological characteristics. But frequencies of tumour-infiltrating lymphocytes varied; in progressing tumours only CD4+ T cells occurred, in temporary regressing tumours also CD8+ T cells were detected. Immune modulators, like interferon alpha or glucocorticoid-induced tumour necrosis factor receptor targeting antibody DTA-1 aggravated the effects of vaccination; latter raised cytotoxic CD8+ T cell numbers and resulted in complete tumour regression. Human HPV+ tumours as well as HPV+ precancerous stages revealed numbers of CD4+ and CD8+ T cells and especially of forkhead box P3+ regulatory T cells that were significantly increased compared to melanoma, bronchial or colon carcinoma. To assist further analysis of human HPV-associated cervical cancer and facilitate studies on therapeutic approaches, a humanized mouse model was established. The present work points to immunological exhaustion in the development of HPV-related tumours comparable to chronic diseases where CD4+ and CD8+ T cells exhaust and immunosuppression by regulatory T cells increases at the same time. For the development of appropriate strategies to enhance efficacy in HPV-associated tumour therapy, further knowledge of mechanisms involved in specific T cell activation, T cell exhaustion and immunosuppression is necessary.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

Cancer is the second leading cause of death in the US and worldwide, accounting for 16% of deaths worldwide in 2015. Of more than 100 types of cancers affecting humans, breast cancer is the most common cancer among women and is the second leading cause of death in women. Triple negative breast cancer (TNBC) is a subtype of breast carcinomas defined by the lack of the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2 /neu). The prognosis and survival of TNBC patients remains the poor due to the lack of effective targeted therapy. Nanotechnology-based drug delivery systems, such as liposomes, are widely investigated to enhance anticancer efficacy by concentrating the drug molecules in the tissues of interest and by altering the pharmacokinetic profile. Taking advantage of the pH gradient in the tumor microenvironment, pH-triggered release is a promising strategy to enhance the anticancer efficacy of drug delivery systems against TNBC. Previously, a strategy in our lab has been developed to render saturated and pegylated liposomes pH-sensitive: protonation-induced conformational switch of lipid tails, using trans-2-aminocyclohexanol lipids (TACH, flipids) as a molecular trigger. Based on previous work in our lab, pH-sensitive liposomes (fliposomes) composed of C-16 flipids with amine group of morpholine (MOR) and azetidine (AZE) demonstrated optimized triggered release in response to the tumor’s low pH microenvironment. In this study, different preparation methods were developed and optimized to produce viable fliposomes with high doxorubicin (DOX) encapsulation efficiency. In vitro release assays were established and validated to accurately reflect pH-triggered release of fliposomes. The physicochemical properties of DOX-loaded fliposomes were characterized and their pH-dependent release were investigated. Factors influencing the desirable attributes of liposomes, such as size, pH-sensitivity, stability and drug-loading capacity were explored. Based on these characterizations, central composite design (CCD) was utilized to optimize the formulation of fliposome with two critical factors, flipids and cholesterol. Cell viability assays on traditional monolayer and innovative three-dimensional multicellular spheroids (3D MCS) of TNBC cell lines were conducted to evaluate the anticancer efficacy of the resultant fliposomes in vitro. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells, as well as acidity inside the 3D MCS based on confocal microscopic imaging studies. The distribution and penetration of DOX-loaded fliposomes into 3D MCS was imaged by confocal microscopy in comparison to DOX-loaded non pH-sensitive liposomes and free DOX. As a result, fliposome manifested superior anticancer activity against TNBC 3D MCS by efficient penetration into 3D MCS, followed by tuning up the release rate of the anticancer agent DOX. A TNBC orthotopic xenograft model was established by transplanting TNBC into the murine mammalian fat pad, which maintains the organ-specific tumor microenvironment of the original organ . A pilot pharmacokinetic study was conducted in order to correlate the pH response and stability properties with the in vivo stability of the optimized AZE-C16 fliposome. The antitumor efficacy was comparable between free DOX and DOX-loaded stealth liposome with tumor volumes of ~ 80-90% of the control treatment 32 days post first dose. In contrast, the DOX-loaded fliposome, especially MOR-C16 fliposome, exhibited a significantly higher antitumor efficacy and delayed progression compared to free DOX and stealth liposome treatments. Taken together, DOX-loaded fliposomes were successfully prepared and optimized for in vivo application. They were able to achieve superior activity against TNBC in vitro and in vivo, facilitated by enhanced release of the anticancer drug DOX after penetration inside TNBC tumor.

Le PCK3145 est un peptide de 15 acides aminés inhibant la sécrétion de MMP-9 et démontrant une activité anti-tumorale contre le cancer de la prostate. Comme les cancers hématologiques sécrètent MMP-9, nous avons donc évalué l’effet du PCK3145 sur ces cancers. Nous avons démontré que les lignées humaines de lymphome non- Hodgkinien (LNH) SR et de myélome multiple RPMI-8226 ainsi que la lignée murine de mastocytome P815 ont une prolifération réduite suite à une exposition au PCK3145. Ce peptide diminue également la clonogénicité de ces cellules. In vivo, le PCK3145 diminue significativement la croissance des tumeurs sous-cutanées P815 comparativement au PBS (p<0.001) et aux peptides contrôles (« scrambled peptide » (p<0.05) et PCK5266 (p<0.01)). De plus, le traitement au PCK3145 diminue le nombre de métastases au niveau du foie par rapports aux contrôles (p<0.05). Les niveaux de MMP-9 dans le sang des souris traitées au PCK3145 sont similaires à ceux dans le sang des souris sans tumeur. Par contre, chez les souris recevant le PBS ou le « scrambled peptide », les niveaux de MMP-9 étaient significativement plus élevés que dans les souris sans tumeur et les souris traitées au PCK3145 (p<0.05). De surcroît, dans un modèle de xénogreffe, le PCK3145 diminue significativement la croissance des lymphomes SR par rapport au PBS (p<0.01) et au « scrambled peptide » (p<0.001). Ces résultats indiquent que le PCK3145 possède une activité anti-tumorale et pourrait représenter un agent intéressant pour le traitement de plusieurs cancers hématologiques. / PCK3145 has been shown to exert anti-tumor activity against prostate cancer cells. In a Phase I clinical study, this peptide demonstrated low toxicity. To determine whether PCK3145 could exert cytotoxic activity against other marrow infiltrating cancers, we tested its activity against hematologic cancers. Interestingly, PCK3145 inhibited the proliferation of human NHL (SR) and myeloma (RPMI-8226) cell lines and murine mastocytoma (P815) cell line in vitro. Moreover, PCK3145 reduced the clonogenicity of these cell lines. To explore its activity in vivo, DBA/2 mice were injected with P815 cells. PCK3145 treatment significantly decreased P815 tumors growth in comparison to PBS (p<0.001), scrambled peptide (p<0.05) and PCK5266 (amino acids 52-66 of PSP-94) (p<0.01). Intraperitoneal PCK3145 treatment led to a decreased number of liver metastasis compared to PBS (p<0.05) and scrambled peptide (p<0.05). MMP-9 levels, measured by ELISA, in the peripheral blood of treated P815 bearing mice were similar to those obtained with healthy animals (12.83 1.890 (mean SD) ng/ml and 6.48 0.4070 ng/ml, respectively), while MMP-9 levels were elevated in mice treated with PBS and scrambled peptide (35.12 8.559 ng/ml and 22.60 3.944 ng/ml, respectively; p<0.05). In NOD/SCID mice PCK3145 treatment resulted in significant inhibition of human NHL SR growth compared to treatment with PBS (p<0.001) and scrambled peptide (p<0.01). Consequently, treatment with PCK3145 can reduce tumor cell proliferation of murine and human hematologic cancers. In addition, PCK3145 has the potential to inhibit tumor cells dissemination by lowering MMP-9 secretion. Thus, PCK3145 represents a unique peptide demonstrating sequence-specific anti-tumor activity hematologic malignancies.

Introdução: O Câncer de próstata metastático (mCaP) é uma doença incurável com progressão para o mCaP resistente à castração (mCPRC) após terapia de deprivação androgênica. Os microRNAs (miR) -23b e -27b tem ação antioncogênica e são suprimidos neste contexto. O gene da ciclina G1 (CCNG1) codifica uma quinase dependente de ciclina com potencial de inibição do crescimento e é um dos alvos dos miR-23b/-27b. Objetivos: Estimular os miR-23b/-27b isoladamente e em conjunto para avaliar e comparar o crescimento tumoral e a expressão do gene alvo CCNG1 em relação ao grupo controle em xenenxertos de PC-3M-luc-C6 em camundongos atímicos castrados. Métodos: Xenoenxertos subcutâneos da linhagem celular PC-3M-luc-C6 foram implantados em camundongos machos BALB/c nude. Os animais foram castrados 10 dias após o implante e utilizamos injeções intratumorais para induzir o aumento da expressão dos miR-23b/-27b separadamente e em conjunto através de Pre-miR® específicos. Realizamos avaliações semanais da bioluminescência (BLI) para avaliar o crescimento tumoral após a castração. Utilizamos a reação em cadeia de polimerase reversa em tempo real (qRT-PCR) para analisar a expressão da CCNG1 e os animais foram sacrificados 21 dias após a castração. Dividimos um total de 21 xenoenxertos nos seguintes grupos de tratamento: 4 no grupo controle, 5 no grupo Pró miR-23b, 6 no grupo Pró miR-27 e 6 no grupo Pró miR-23b associado ao Pró miR-27b. Resultados: Confirmamos o sucesso da transfecção dos miRs por qRT-PCR, e apresentamos o achado de superexpressão relativa da CCNG1 em relação ao grupo controle em: 9% (p=0,76), 46% (p=0,05) e 203% (p=0,01) nos grupos Pró miR-23b, Pró miR-27b e Pró miR-23b associado ao Pró miR-27b respectivamente. Comparamos o crescimento proporcional de cada tumor através da BLI, por meio da leitura no momento da castração ao final do experimento. Obtivemos um crescimento de 13,5; 8,69; 5,96 e 9,98 vezes nos grupos: controle, Pró miR-23b, Pró miR-27b e Pró miR-23b associado ao Pró miR-27b respectivamente. Conclusão: Demonstramos um modelo in vivo de CPRC que apresentou supreexpressão da CCNG1 após o tratamento intratumoral que aumentou a expressão dos miRs -23b e -27b. Este conjunto de miRs tem ação antioncogênica descrita no contexto do mCPRC e a sua estimulação neste contexto aumentou a expressão da CCNG1. Nosso estudo sugere que a CCNG1 deve apresentar uma ação pró-apoptótica quando superexpresso pelos miRs-23b/-27 no CPRC / Introduction: Metastatic prostate cancer (mPCa) is an incurable disease that invariably progresses to castration-resistant mPCa (mCRPC) after androgen deprivation therapy. The microRNAs miR-23b/-27b have been reported as tumor suppressors and are underexpressed in this context. The cyclin G1 gene (CCNG1) encodes a cyclin-dependent kinase with potential growth inhibitory activity that is a potential target of miR-23b/-27b. Objectives: We aim to explore a bioluminescent xenograft model of CRPC in castrated mice the effect positive modulation of the miR-23b/-27b on CCNG1 expression and mCRPC growth. Material and Methods: We injected subcutaneous xenografts of PC-3M-luc-C6 PCa cell line in BALB/c nude male mice. We neutered the animals after 10 days and used intratumoral injections up-regulating miR-23b/-27b separately and simultaneously through specific Pre-miRTM. We used weekly bioluminescence imaging (BLI) to assess tumor growth after castration and real-time polymerase chain reaction (qRT-PCR) to analyze the expression of CCNG1. We sacrificed the animals 21 days after castration. We randomized 21 xenografts in experimental groups as follows: n=4 in the negative control group; n=5 in Pro miR-23b group; n=6 in Pro miR-27b group and an n=6 tumors in the Pro miR-23b plus Pro miR-27b. Results: We confirmed successful transfection of both miRNAs with overexpression of CCNG1 of 9% (p=0.76), 46% (p=0.05) and 203% (p=0.01) in the Pro miR-23b, Pro miR-27b and Pro miR-23b plus -27b groups respectively. We compared the fold-change in BLI growth by the end of experiment finding an increase of 13.5-fold, 8.69-fold, 5.96-fold and 9.98-fold in groups Pro miR-negative control, Pro miR-23b, Pro miR-27b and Pro miR-23b plus Pro miR-27b groups respectively. Conclusions: We showed an in vivo model with overexpression of CCNG1 upon artificial upregulation of miR-23b and -27b in CRPC. This cluster of antineoplastic miRNA increased the expression of this cyclin, often described as oncogenic. Our study suggests that CCNG1 has a pro-apoptotic role when up-regulated by miR-23b/-27b in CPRC

Le PCK3145 est un peptide de 15 acides aminés inhibant la sécrétion de MMP-9 et démontrant une activité anti-tumorale contre le cancer de la prostate. Comme les cancers hématologiques sécrètent MMP-9, nous avons donc évalué l’effet du PCK3145 sur ces cancers. Nous avons démontré que les lignées humaines de lymphome non- Hodgkinien (LNH) SR et de myélome multiple RPMI-8226 ainsi que la lignée murine de mastocytome P815 ont une prolifération réduite suite à une exposition au PCK3145. Ce peptide diminue également la clonogénicité de ces cellules. In vivo, le PCK3145 diminue significativement la croissance des tumeurs sous-cutanées P815 comparativement au PBS (p<0.001) et aux peptides contrôles (« scrambled peptide » (p<0.05) et PCK5266 (p<0.01)). De plus, le traitement au PCK3145 diminue le nombre de métastases au niveau du foie par rapports aux contrôles (p<0.05). Les niveaux de MMP-9 dans le sang des souris traitées au PCK3145 sont similaires à ceux dans le sang des souris sans tumeur. Par contre, chez les souris recevant le PBS ou le « scrambled peptide », les niveaux de MMP-9 étaient significativement plus élevés que dans les souris sans tumeur et les souris traitées au PCK3145 (p<0.05). De surcroît, dans un modèle de xénogreffe, le PCK3145 diminue significativement la croissance des lymphomes SR par rapport au PBS (p<0.01) et au « scrambled peptide » (p<0.001). Ces résultats indiquent que le PCK3145 possède une activité anti-tumorale et pourrait représenter un agent intéressant pour le traitement de plusieurs cancers hématologiques. / PCK3145 has been shown to exert anti-tumor activity against prostate cancer cells. In a Phase I clinical study, this peptide demonstrated low toxicity. To determine whether PCK3145 could exert cytotoxic activity against other marrow infiltrating cancers, we tested its activity against hematologic cancers. Interestingly, PCK3145 inhibited the proliferation of human NHL (SR) and myeloma (RPMI-8226) cell lines and murine mastocytoma (P815) cell line in vitro. Moreover, PCK3145 reduced the clonogenicity of these cell lines. To explore its activity in vivo, DBA/2 mice were injected with P815 cells. PCK3145 treatment significantly decreased P815 tumors growth in comparison to PBS (p<0.001), scrambled peptide (p<0.05) and PCK5266 (amino acids 52-66 of PSP-94) (p<0.01). Intraperitoneal PCK3145 treatment led to a decreased number of liver metastasis compared to PBS (p<0.05) and scrambled peptide (p<0.05). MMP-9 levels, measured by ELISA, in the peripheral blood of treated P815 bearing mice were similar to those obtained with healthy animals (12.83 1.890 (mean SD) ng/ml and 6.48 0.4070 ng/ml, respectively), while MMP-9 levels were elevated in mice treated with PBS and scrambled peptide (35.12 8.559 ng/ml and 22.60 3.944 ng/ml, respectively; p<0.05). In NOD/SCID mice PCK3145 treatment resulted in significant inhibition of human NHL SR growth compared to treatment with PBS (p<0.001) and scrambled peptide (p<0.01). Consequently, treatment with PCK3145 can reduce tumor cell proliferation of murine and human hematologic cancers. In addition, PCK3145 has the potential to inhibit tumor cells dissemination by lowering MMP-9 secretion. Thus, PCK3145 represents a unique peptide demonstrating sequence-specific anti-tumor activity hematologic malignancies.

BACKGROUND: Activating mutations of FGFR3 are frequently identified in superficial urothelial carcinoma (UC) and increased expression of FGFR1 and FGFR3 are common in both superficial and invasive UC. METHODS: The effects of inhibition of receptor activity by three small molecule inhibitors (PD173074, TKI-258 and SU5402) were investigated in a panel of bladder tumour cell lines with known FGFR expression levels and FGFR3 mutation status. RESULTS: All inhibitors prevented activation of FGFR3, and inhibited downstream MAPK pathway signalling. Response was related to FGFR3 and/or FGFR1 expression levels. Cell lines with the highest levels of FGFR expression showed the greatest response and little or no effect was measured in normal human urothelial cells or in UC cell lines with activating RAS gene mutations. In sensitive cell lines, the drugs induced cell cycle arrest and/or apoptosis. IC(50) values for PD173074 and TKI-258 were in the nanomolar concentration range compared with micromolar concentrations for SU5402. PD173074 showed the greatest effects in vitro and in vivo significantly delayed the growth of subcutaneous bladder tumour xenografts. CONCLUSION: These results indicate that inhibition of FGFR1 and wild-type or mutant FGFR3 may represent a useful therapeutic approach in patients with both non-muscle invasive and muscle invasive UC.