Carl Spickett
Academic Laboratory of Medical Genetics Academic Laboratory of Medical Genetics Academic Laboratory of Medical Genetics Academic Laboratory of Medical Genetics
Human Karyotype Human Karyotype Human Karyotype Human Karyotype "NHGRI human karyotype" by Courtesy: National Human Genome Research Institute
Genes to Proteins Genes to Proteins Genes to Proteins Genes to Proteins
Von Hippel Von Hippel Von Hippel Von Hippel- - - -Lindau Disease Lindau Disease Lindau Disease Lindau Disease Rare inherited cancer syndrome named after Eugen Von Hippel and Arvid Lindau who described retinal angiomatosis and spinal haemangiobastomas respectively. Autosomal Dominant Inheritance from either parent Caused by VHL gene mutation on Chromosome 3 Loss of function of pVHL protein
Von Hippel Lindau Disease Von Hippel Lindau Disease Von Hippel Lindau Disease Von Hippel Lindau Disease Spinal Cerebella Haemangioblastoma Haemangioblastoma Retinal Angiomas Adrenal gland Pheochromocytoma Pancreatic Cysts Renal Cysts and Renal Cell Carcinoma
HLRCC HLRCC HLRCC HLRCC • Hereditary Leiomyomatosis and Renal Cell Carcinoma • Rare kidney cancer susceptibility syndrome (Type 2 Papillary RCC) • Caused by mutations in FH gene encoding Fumerate Hydratase • Like VHL it is inherited in an autosomal dominant fashion • Benign growths present on skin (Leiomyomas)
Tumour Suppressor Gene (TSG) Tumour Suppressor Gene (TSG) Tumour Suppressor Gene (TSG) Tumour Suppressor Gene (TSG) Cell division Cell growth (mitosis) Cell Cycle Synthesis of new DNA Tumour suppressor Gene (TSG)
VHL VHL VHL VHL – – – – The story of a TSG The story of a TSG The story of a TSG The story of a TSG L. Gossage et al., 2015. Nature Reviews Cancer
VHL Tumour Suppressor Gene VHL Tumour Suppressor Gene VHL Tumour Suppressor Gene VHL Tumour Suppressor Gene Adapted from: KEGG Pathways (clear-cell RCC)
Kidney Cancer Kidney Cancer Kidney Cancer Kidney Cancer • Kidney cancers are eighth most common cancer type • RCC incidence has steadily increased in last 30 years • Early detection and removal CAN be curative, however poor prognosis for metastatic disease. Targeted lethality of cells deficient in RCC gene function will enable advances in treatment for both inherited and sporadic RCC.
Clear Cell Renal Cell Carcinoma ( Clear Cell Renal Cell Carcinoma ( Clear Cell Renal Cell Carcinoma ( Clear Cell Renal Cell Carcinoma (ccRCC ccRCC ccRCC ccRCC) ) ) ) o Most common form of kidney cancer (~75 %) o Mutations in VHL TSG most common genetic event o Small fraction of RCC is accounted for by rare inherited monogenic disorders: • von Hippel-Lindau disease ( VHL TSG encoding pVHL) • Birt-Hogg-Dubé syndrome ( FLCN TSG encoding folliculin) • Hereditary leiomyomatosis renal cell cancer (HLRCC) ( FH encoding fumerate hydratase) • Hereditary papillary RCC ( MET proto-oncogene encoding hepatocyte growth factor receptor (HGFR)) • Succinate dehydrogenase subunit disorders (SDHB, SDHD)
Knudson’s ‘Two hit’ model Knudson’s ‘Two hit’ model Knudson’s ‘Two hit’ model Knudson’s ‘Two hit’ model Familial mutation Sporadic cancer
Clear Cell Renal Cell Carcinoma mutations Clear Cell Renal Cell Carcinoma mutations Clear Cell Renal Cell Carcinoma mutations Clear Cell Renal Cell Carcinoma mutations 60 VHL 50 40 PBRM1 30 20 SETD2 BAP1 10 KDM5C mTOR TP53 PTEN PIK3CA TCEB1 0 Adapted from: Molecular Genetics of Clear-Cell Renal Cell Carcinoma. James Brugarolas. 2014.
Existing Therapies Existing Therapies Existing Therapies Existing Therapies Treatment Type Target Kinase Inhibitors Sorafenib Multikinase Inhibitor Inhibitor of Ras signalling, VEGF receptors and PDGF receptors. Sunitinib Receptor tyrosine kinase (RTK) Inhibitor Inhibitor of VEGFR, PDGFR and c-Kit (CD117) signalling Axitinib and VEGFR Tyrosine kinase inhibitor Inhibits downstream targets of VEGFR Tivozanib Dovitinib Oral FGFR and VEGFR inhibitor Inhibits downstream targets of VEGFR and FGFR Pazopanib Oral Multikinase angiogenesis inhibitor Inhibits VEGFR, PDGFR and c-Kit signalling mTOR Inhibitors Temsirolimus mTOR inhibitor (stimulator of ribosomal mTOR inhibition decreases HIF1α translation translation of multiple mRNAs) Everolimus Oral mTOR inhibitor Used after failed treatment with Sorafenib or Sunitinib Antibodies Bavacizumab VEGF monoclonal antibody (in Targets and inactivates VEGF (HIF target) for treatment of conjunction with IFNα) advanced ccRCC. IFNα to increase immune response.
Existing Therapies Existing Therapies Existing Therapies Existing Therapies Kinase Inhibitors Dovitanib Tivozanib Sorafenib Sunitinib Temsirolimus Axitinib Pazopanib Everolimus mTOR inhibitors Bavacizumab Adapted from: KEGG Pathways (clear-cell RCC)
Cell Lines Cell Lines Cell Lines Cell Lines • We have 21 cell lines at our disposal! • Cell lines with and without functional pVHL • Cells have been put through ‘Trusight cancer panel’ to check for cancer associated changes • Very useful for high-throughput assays and target identification • Cells in culture are relatively amenable to alteration 1. Knock-down 2. Knock-out 3. Rescue
Synthetic Lethality Synthetic Lethality Synthetic Lethality Synthetic Lethality Synthetic lethality in cells not expressing pVHL Survival in cells stably transfected for VHL (rescue) 25 kDa pVHL Beta-actin
Cells in Culture Cells in Culture Cells in Culture Cells in Culture
Cells in Culture Cells in Culture Cells in Culture Cells in Culture
Cell Screening Cell Screening Cell Screening Cell Screening
What Next? What Next? What Next? What Next? 0 hours 17 hours 24 hours Wound healing ‘scratch assay’ Colony formation assays Cell migration assays
3 3 3 3- - - -D Culture D Culture D Culture D Culture • Cells appear to form spherical structures in 3D culture • These spheroids are organised ball-like shapes • Could be used as a qualitative indicator for cell dysfunction • Show effect of depletion of target protein on cell growth
Drug Development Drug Development Drug Development Drug Development
Identification of Therapeutic Agents Identification of Therapeutic Agents Identification of Therapeutic Agents Identification of Therapeutic Agents The ‘Pfizer Three Pillars’ Drug must be exposed at the target site Must bind pharmacological target as expected mode of action Must show pharmacological activity and illicit a response
Summary Summary Summary Summary Cell biology approach to target identification for drug development Challenging work which requires patience, persistence and positivity We are a long way from developing new treatments but we are working hard and forming important collaborations Realistic project aim is target identification and testing in animal disease models (mice and zebrafish)
Thank you to: Group Leader Eamonn Maher Lab members France Docquier Hilda Mujcic Hannah West Phil Smith Benoit Lan-Leung Graeme Clarke Ezequiel Rodriquez Eguzkine Ochoa Ruth Casey James Whitworth
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