BMS3031 Exam.pdf

Learning outcomes 1. Explain the pathophysiology of Hunter Syndrome 2. Evaluate whether enzyme replacement therapy (ERT) for Hunter syndrome has been successful 3. Explain how adeno-associated virus (AAV) vectors can be used to deliver gene therapy treatments Image source: https://www.skillshub.com/blog/learning-outcomes/ 4. Determine the mechanism through which the IDS gene is inserted into the patients genome during gene therapy treatment Gene therapy which Gene therapy that introduces an episome changes the patients DNA DOES NOT alter the patients own Often called “gene editing” genome/genetic material Requires a nuclease to cut the genome (Zinc Fingers, TALENs, CRISPR-Cas9) Genetic material delivered is in the form of an episome (in the nucleus) that remains Once genome is cut, desired DNA can be separate from the genome inserted using homology directed repair (HDR) No nuclease required (genome is not cut) Note – Non-homologous end joining (NHEJ) won’t result in insertion of desired DNA/gene Episome Gene therapy which Gene therapy that introduces an episome changes the patients DNA DNA changes made via gene editing Episomes are not passed onto daughter are passed onto the daughter cells cells originating from the cell that derived from the “edited” cell contains the episome Episome Gene therapy - Usually involves viral mediated introduction of defective cells with corrective gene - ADA (adenosine deaminase) defect in SCID corrected in T cells by retroviral gene therapy but stopped in 2002 due to development of leukemia - AAV now the choice of expression system (r)AAV: (recombinant) adeno-associated virus AAV = single stranded, DNA virus - has a "simple" genome packaged in an icosahedral capsid (protein shell). - For biotech, the genome is typically gutted so that precious cargo space is for gene delivery, and for safety. - Size of insertion is limiting factor - AAV vectors lack the integration- promoting gene and therefore only rarely and randomly integrate into the human genome. - rAAV can transduce both dividing and non-dividing cells, with stable transgene expression for years in postmitotic tissue. - Not pathogenic and very low November 2017: 204 clinical trials immunogenicity worldwide have used AAV vectors rAAV transduction pathway translation transcription RPE65 gene therapy for Leber congenital amaurosis A common causes of blindness in children (2-3 per 100,000 newborns) Autosomal recessive mutations in about 13 different genes including RPE65 RPE65 produced in retinal pigment epithelium (RPE) and is required in the visual cycle VISUAL CYCLE: Absorption of light causes isomerization of 11- cis-retinal to all-trans-retinal in photoreceptors for phototransduction Decay of activated rhodopsin yields opsin and all-trans-retinal, which is reduced to all-trans- retinol. All-trans-retinol diffuses into the RPE where it is converted back to 11-cis-retinal in a reaction involving RPE65. 11-cis-retinal, which then diffuses back into the photoreceptor where it combines with opsin to regenerate visual pigments. Luxturna: to correct RPE65 deficiency Dec 19, 2017: The first in vivo gene therapy approved by the FDA (made by Spark Therapeutics) $850K per patient! (but effective) Spark Therapeutics, bought out by Roche $4.8bn in 2019! Did you know? Elaprase is an enzyme replacement therapy for Hunter syndrome Not of the PBS in Australia Modifying genes following the introduction of double strand breaks NON-HOMOLOGOUS END JOINING (NHEJ) HOMOLOGOUS RECOMBINATION (HR) Intrinsically error prone & results in formation Requires the addition of template DNA of indels (small insertions/deletions of with homologous arms but can have typically 5- 20 nucleotides). variable sequences (mutations, Can disrupt reading frame of genes if targeted insertions) in between. appropriately Types of therapeutic gene editing Gene disruption: Silence a pathogenic gene NHEJ gene correction: Deletion of a pathogenic insertion HDR gene correction: Correct a deleterious insertion HDR= homology directed repair Targeting specific genes using site directed nucleases Zinc finger nucleases (ZFNs) Series of protein modules that bind 3 nucleotides bringing Fok1 nuclease to site (dimer becomes active) Transcription activator-like effector nucleases (TALENs) Series of protein modules that bind individual nucleotides bringing Fok1 nuclease to site (dimer becomes active) RNA-guided engineered nucleases (CRISPR/Cas9) RNA molecule PAM protospacer (CRISPR) designed that binds nucleotides and targets Cas9 nuclease to site Activity 1 – Enzyme replacement therapy ~25 Minutes 1. Describe the pathophysiology of Hunter Syndrome using the diagram below as a guide. 2. Explain how Elaprase (Idursulfase) is produced for Enzyme Replacement Therapy (ERT). ACTIVITY 1 3. Illustrate the mechanism of action of Elaprase in Hunter Syndrome. Mechanism of Action of POC tests 4. Describe the clinical success and limitations of Elaprase. See resourced needed for workshop activities.doc on week 3 tab of Moodle r which cannot be effectively remodelled Hunter pathophysiology continued… The chronic progressive nature of Hunter syndrome s caused by the accumulation of partially degraded GAGs, with resulting thickening of tissue and compromising of cell and organ function over time. - e.g. thickening of skin & organs IDS (iduronate sulfatase) gene produces the I2S protein (iduronate 2-sulfatase) Incorrect glycosylation of the I2S protein may interfere with lysosomal targeting through by impaired binding to the mannose-6- phosphate receptor. Active site binds to part of the GAG to remove a sulfate group to facilitate the breakdown of the GAGs Insights into Hunter Syndrome from the structure of iduronate-2- Sulfatase Demydchuk et al., 2017 Nature Communication Many different types of mutations in the IDS gene cause Hunter syndrome “Although enzyme replacement therapy (ERT) with idursulfase has been shown to improve somatic signs and symptoms of Hunter syndrome, the drug does not cross the blood–brain barrier and so treatment of neurological aspects of the disease remains challenging.” Whiteman & Kimura (2017) Intracerebroventricular injection an option but poses difficulties for widespread use Does Elaprase work in practice? Lower amount of creatinine Does Elaprase work in practice? in urine indicated improved kidney health All viral genes are removed from AAVs when used for gene therapy or gene editing Rep genes - No chance that viral genes will insert into a patients Cap genes genome Rep genes: Cap genes: - transcription, replication & - Involved in the formation of the viral packaging of viral genetic material capsid that forms around the viral DNA Activity 2 – Gene replacement therapy ~20 Minutes Illustrate the mechanism of action of gene therapy in Hunter Syndrome, including: ACTIVITY 1 Enzyme 1. replacement The production therapy of AAVs containing the IDS gene 2. How AAVs deliver IDS gene to its target 3. The mechanism of the IDS gene insertion into Image source: https://altoida.com/blog/gene-therapy-for- alzheimers-disease-understanding-the-research/ the target locus See resourced needed for workshop activities.doc on week 3 tab of Moodle AAV gene therapy for Hunter Syndrome Deliver 3 different AAVs to a liver cell: Zinc finger proteins 1 & Zinc finger proteins 2 bind to a specific part - 1 IDS gene AAV need to be transcribed of the albumin gene to - 1 Zinc Finger no. 1 DNA & translated in the introduce a double instructions liver cell stranded break - 1 Zinc Finger no. 2 DNA instructions Homology direct repair (HDR) ? ? closes the double stranded Increased amount of break using the IDS gene with Reduction of Hunter I2S enzyme made in complementary regions syndrome symptoms in the the liver cells from liver, maybe also in other IDS gene added by = functional IDS gene is now organs gene therapy within the albumin gene Constructing AAVs in vitro Collect AAVs that have all Need to constructive 3 different AAVs: three plasmids - 1 for IDS gene - 1 for ZF1 - 1 for ZF2 Gene therapy to treat Hunter syndrome delivered to the liver mRNA transcripts Miller et al 2007 VOLUME 25 NUMBER 7 JULY 2007 NATURE BIOTECHNOLOGY Comparison of different platforms Zn finger nucleases TALENs CRISPR/Cas9 Impact on patients Once IDS (iduronate sulfatase) is inserted in the albumin locus, hepatocytes express the I2S (iduronate 2-sulfatase) protein. In theory, if with a large amount of the I2S protein is being produced in the liver from the transgene that has been added, it should be able to facilitate breakdown of GAGs in the lysosome in the hepatocyte cells.  This would in theory, lessen the burden of Hunter syndrome on the hepatocyte cells. Preliminary results (2018) ▪ Urine GAG levels in Brian Madeux and another low-dose patient rose 9% on average after 4 months ▪ Two other patients were given a middle dose that was twice what the first two patients received ▪ Their GAG levels declined by 51% after 4 months, on average ▪ Blood tests did not detect the missing enzyme - could be because any that was being made was rapidly used by cells rather than getting into the bloodstream Lentivirus gene therapy approach for Hunter syndrome now underway! https://www.manchester.ac.uk/discover/news/groundbreaking-gene- therapy-trial-for-hunter-syndrome-opens/ Inteins to split Cas9 for packaging into AAVs Inteins – “protein introns” that splice out autocatalytically from host polypeptides to generate a functional protein. The N-terminal half of Cas9 is fused upstream of the N-intein and packaged in one AAV and the C-terminal half of Cas9 fused downstream of the C-intein is packaged into another AAV. When expressed together, the inteins undergo post-translational autocatalytic excision while ligating the two Cas9 portions together Can we quantify drug distribution? Volume of distribution if a drug does not readily move out of the plasma compartment (e.g. very large molecule/high degree of binding to plasma proteins) volume it is distributed in is ~ plasma volume if a drug very easily moves out of the plasma compartment (e.g. very lipophilic molecule) volume it is distributed in is >>>plasma volume estimate distribution by comparing amount in the body (ie dose) to the plasma concentration Volume of distribution (Vd) A theoretical concept: = volume into which a drug appears to be distributed with a concentration equal to that of plasma relates the plasma concentration to the total amount of drug in the body Vd = total amount of drug in the body / [plasma] Vd = dose / [plasma] units volume (or volume / kg body weight) https://www.icp.org.nz/volume-of-distribution/simple-container-model Volume of distribution Volume In a 70 kg person Plasma volume 0.05 L/kg ~3.5 L Total extracellular volume 0.2 L/kg ~14 L Total body water 0.55 L/kg ~40 L Vd ~0.05 L/kg body wt – drug retained in vascular ‘compartment’ (eg high mw; extensive protein binding) Vd ~0.2 L/kg body wgt – drug restricted to extracellular fluid (eg low MW but hydrophillic) Vd ~0.55 L/kg body wgt – drug distributed throughout TBW (eg very lipophilic, digoxin) metabolic degradation renal excretion of inactive drug renal excretion of hepatobiliary unchanged active excretion of drug inactive drug Renal Clearance glomerular filtration ( urine) depends on [free drug] in plasma & GFR tubular secretion ( urine) e.g. penicillin (via OAT); morphine (via OCT) reabsorption ( plasma) depends on lipid solubility & urine flow rate http://en.wikipedia.org/wiki/Clearance_(medicine) Total amount eliminated via kidneys = amount filtered + amount secreted – amount reabsorbed Predict the difference in renal clearance between a very lipophilic drug a highly ionised drug a biological (ie large molecule) Drug metabolism makes lipid soluble drugs more water soluble so that they can be excreted via the kidneys depends on many different types of enzymes mostly located intra-cellularly http://www.dolcera.com/wiki/index.php?title=Drug_Metabolism Enzymatic metabolism of drugs Phase I reactions Phase II reactions – CATABOLIC – ANABOLIC “Functionalisation” “Conjugation” i.e. oxidation, reduction or combined with endogenous hydrolysis   polarity molecule (e.g. glucuronyl, glycyl, glutathione, acetyl, sulphate, methyl group)  more water-soluble  more water-soluble reactions may be sequential = Phase II = Phase I Diagram taken from Lubel et al., Med J Aust 2007; 186 (7): 371-372 Phase II drug metabolism occurs mainly in the liver metabolite is: pharmacologically inactive (usually) less lipid-soluble excreted in urine or into bile (faeces) potentially saturable not considered important for drug-drug interactions reactions may be sequential = Phase II = Phase I Diagram taken from Lubel et al., Med J Aust 2007; 186 (7): 371-372 Phase I drug metabolism occurs in liver, but also in other tissues (e.g. intestine) metabolites maybe more active / toxic: e.g. paracetamol (metabolite  hepatocellular death) prodrugs (e.g. codeine  morphine) potentially saturable potential site for drug-drug interactions Phase I drug metabolising enzymes broad substrate specificity 1 enzyme may catalyse the metabolism of many different drugs 1 drug may be metabolised by multiple enzymes / isoenzymes liver drug metabolising enzymes usually embedded in endoplasmic reticulum (microsomal) important family = cytochrome P450 “super” family catalyse oxidative metabolism of a range of xenobiotics cytochrome P450s differ in: amino acid sequence (coding genes) sensitivity to inhibitors & inducing agents substrate specificity enzyme reaction Fig 9.2 Rang et al (2016) requires: molecular oxygen, NADPH; NADPH–P450 reductase (a flavoprotein) forms a hydroxylated product ~74 CYP450 gene families three major families involved in drug metabolism in liver (CYP1, CYP2, CYP3) Caution when elimination involves enzymic metabolism Metabolising enzymes: are potentially saturable ….. activity can be influenced by many factors including: genetics other drugs drug interaction via metabolising enzymes Enzyme inhibition drugs metabolised by the same enzyme, if administered together will compete for the metabolising enzyme Enzyme induction some drugs can  activity/levels of the enzymes that metabolise them (e.g. alcohol, barbiturates) interaction can also be between drugs and “herbal” medicines (e.g. St Johns Wort) or food components (e.g. grapefruit juice) Biliary clearance of drugs  Enterohepatic recycling Other routes for excretion of drugs … lungs can remove gases and volatile liquids (e.g. many general anaesthetics) sweat, saliva, mucous, tears & milk can excrete small amounts of drugs (some drugs in milk can be dangerous to infants) Polymorphisms in cytochrome P-450 all genes encoding CYP 1/2/3 family of P-450 enzymes are polymorphic clinically important polymorphism is seen in CYP2C9 / CYP2C19 CYP2D6 CYP3A4 (account for 60-70% phase 1 dependent metabolism of clinically important drugs) Variation in CYP alleles e.g. CYP2D6 – metabolises 25% drugs Consequences of polymorphisms in CYP2D6 20-30 million subjects have 15-20 million subjects have no CYP2D6 enzymes CYP2D6 gene duplications ( poor metabolisers) (ultrafast metabolisers) drug metabolism too slow drug metabolism too fast drug levels too high at no drug response at ordinary ordinary dose dosage (non-responders) high risk for adverse effects no response from specific prodrugs Warfarin equal mixture of S and R enantiomers commonly prescribed (2 million per year in the US) high rate of adverse events warfarin maintenance doses are characterized by large inter-individual variability maintenance doses can range 50-fold (eg, daily dose requirements range from 0.5 to 25 mg) Warfarin pharmacogenetics …. metabolism of warfarin occurs through CYP2C9 clinically relevant SNPs have been identified in CYP2C9 these result in reduced enzymatic activity  metabolism even after adjusting the warfarin dose for the variability in CYP2C9 status, there is still an amount of dosing variability in patients who have similar CYP2C9 alleles additional variability is due to polymorphisms in the warfarin target, vitamin K reductase complex (VKORC1) Pharmacogenomics the use of genetic information to guide drug therapy  differences in the response of individuals to drugs can be predicted from knowledge of genetic make up  need to get drug to the target site at the appropriate [ ] and for the appropriate amount of time plasma [drug] minimum effective concentration (MEC) time Plasma [drug] (Cp) with drug administration Constant infusion Repeat dosing Plasma [drug] Plasma [drug] Time Time Cp = Dose Rate (DR) Clearance Drug Clearance… Clearance (CL) = the volume of plasma cleared of drug per unit time (units = volume / time) CL (total) = CL(renal) + CL(liver) + CL(other) depends on: depends on: [drug] in urine hepatic blood flow urine flow rate intrinsic clearance [free drug] in plasma [free drug] in plasma How long does it Plasma [drug] (Cp) after stopping… take to eliminate the drug? plasma [drug] Z time Quantifying drug elimination… plasma half-life (t½) = time taken for the amount of drug in the plasma to fall by half major determinant of: dosing frequency time to eliminate the drug (4-5 t½s) time required to reach steady state with repeat dosing (4-5 t½s) For most drugs: t½ is constant t½ = 0.693 x Vd CL t½ and steady state Cp = Dose Rate (DR) Clearance dosage interval doesn’t affect mean steady state [ ] achieved or rate at which it is achieved (~4xt½’s) (as long as it is taken at the half-life) 1st order elimination kinetics Rate of elimination is driven by Cp Constant/repeated dosing will achieve a steady state Cp Time to reach steady state is determined by t½ Time for “removal” is determined by t½ BUT….. for some drugs (e.g. alcohol, aspirin) Rate of elimination independent of Cp plasma half-life (t½) DRUGS WITH ZERO ORDER KINETICS – t½ will vary with plasma [ ] relatively small changes in dose can lead to disproportionate increase in plasma concentration!! (first order) (zero order) Putting it together …… Dosing Rate of elimination = CL x Cp at steady state : dose in = dose out (RE) Maintenance dose = dose rate equivalent to RE ie Maintenance dose rate (DR) = RE or DR = CL x target Cp Consider ….. Drug t ½ (hours) time to steady state flucytosine 4.2 ~16.8 hours digoxin 40.0 ~160 hours chloroquine 200.0 ~5 weeks Putting it all together …… Dosing Maintenance dose rate (DR) must = RE ie DR = CL x target [ ] Loading dose (LD) Vd = dose / [plasma] LD = Vd x target Cp Patient Presentation A 35 year old female patient presents to the GP with the following symptoms: -Fatigue - Joint pain -‘butterfly’ rash Image source: https://kymeramedical.com/tag/butterfly-rash/ 1. Based on these symptoms, which autoimmune disease is the patient likely to have? 2. In order to confirm your suspicions about this autoimmune disease, what test(s) would you run to diagnose the patient? Test Results After receiving a kidney biopsy and immunohistology testing was performed on the glomerulus: Glomerulus showing global mesangial and endocapillary Immunofluorescence staining for IgG showing abundant IgG proliferation deposition in a glomerulus. 1. Can you explain to the patient how the immune-complexes seen in the immunofluorescent image above were generated & how they can cause nephritis? AUTOANTIBODY-INDUCED PATHOGENESIS A multitude of pathophysiological pathways acantholysis Examples of autoantibody-mediated pathogenesis: 1. Mimic hormone stimulation of receptor 2. Blockade of neural transmission by receptor blockade or alteration of the synaptic structures 3. Triggering uncontrolled microthrombosis 4. Induction of altered signaling 5. Uncontrolled neutrophil activation 6. Cell lysis 7. Induction of inflammation at the site of autoantibody binding Nimmerjahn et al, Front. Immunol., 2017 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Etiology of SLE: A mixture of genetics and environment A systemic disease Mostly in women (Female : male = 9:1) High morbidity Different epidemiologic subgroups (eg, race/ethnicity, gender, and age of onset) tend to have varying degrees of disease activity and may thus affect disease outcome SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Disease manifestations in SLE Lupus malar ‘butterfly’ rash Arthritis CNS inflammation (myelitis) SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Lupus nephritis: Deposition of immune complexes in kidneys glomeruli Control glomeruli Glomerulus showing global mesangial and Immunofluorescence staining for IgG showing abundant endocapillary proliferation. Numerous infiltrating IgG deposition in a glomerulus. The presence of these leukocytes can be seen filling the glomerular immune deposits recruits inflammatory cells and causes capillaries. Glomerular basement membranes injury to the glomerulus. appear thickened owing to the presence of immune deposits on the inside and outside surfaces of the glomerular capillaries https://consultqd.clevelandclinic.org/ SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Mortality in SLE is linked to early organ damage Survival probability in patients with and without early damage 1.0 Initial SDI=0 No early organ damage Survival Probability 0.9 (n=190) 0.8 0.7 Initial SDI >0 Evidence of early organ damage (n=73) 0.6 0 2 4 6 8 10 Disease Duration (y) Rahman P et al. Lupus. 2001;10(2):93-96. SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Autoantibodies in SLE are against molecules in nuclei Autoantigens in SLE: dsDNA, ribonucleoproteins, other nuclear proteins complexed with nucleic acids. These are ligands for ‘danger sensors’ – e.g. TLR7 and TLR9 - on antigen presenting cells and B cells SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Cyclic exacerbation of disease Liu & Davidson 2012. Nature Medicine 18:871 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Genetic predisposition Clearance of apoptotic particles and The role of SLE risk alleles in the pathogenesis of SLE immune complexes Polymorphisms in genes involved in the immune Innate immunity clearance of apoptotic particles and nucleic-acid– containing immune complexes (orange) may induce the enhanced activation of pDCs and autoreactive B cells, leading to the production of type I interferon (IFN) and the expansion of autoreactive effector cells, respectively. Polymorphisms in genes involved in innate immunity (green) regulate the induction of, as well as the response to, type I IFN. Abnormal function of innate immune cells activates the adaptive immune system. Both the innate and adaptive immune systems contribute to the inflammatory response and tissue damage (purple). A third major group of polymorphic genes is involved in Adaptive immunity ligand recognition, receptor signaling and other Tissue damage immunological functions of adaptive immune cells (blue). Liu & Davidson 2012. Nature Medicine 18:871 SYSTEMIC LUPUS ERYTHEMATOSUS Examples of genes causing monogenic Genetic predisposition forms of SLE or SLE-like syndromes SLE has >100 disease-associated alleles (A20) RA and IBD have about 120 Crohn’s disease has about 110 The proportion of phenotypic variances explained by variants in human leukocyte antigen (HLA) is 2.6%. Other SLE-associated allele examples: TNFAIP3 Loss of function → enhanced TNF signaling (inflammation) TLR7 gain of function → enhanced MyD88 signalling (B cell activation/inflammation) Villalvazo P. et al, Clinical Kidney Journal, 2022 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Genetic predisposition: Toll-like receptor (TLR) gene variants TLR7 gain-of-function mutation variant: TLR7Y264H o TLR7 is a sensor of viral RNA and binds to guanosine o Extrafollicular origin of pathogenic B cells o MyD88 (an adaptor protein downstream of TLR7) dependent Highly conserved across species Making mouse model to validate CRISPR Brown G.J. et al, Nature, 2022 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Genetic predisposition: Toll-like receptor (TLR) gene variants TLR7 gain-of-function mutation variant: TLR7Y264H o TLR7 is a sensor of viral RNA and binds to guanosine o Extrafollicular origin of pathogenic B cells o MyD88 (an adaptor protein downstream of TLR7) dependent SLE-like phenotypes in TLR7Y264H mouse model (kik/kik) ANA Proliferative glomerulonephritis Brown G.J. et al, Nature, 2022 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Cyclic exacerbation of disease Liu & Davidson 2012. Nature Medicine 18:871 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Environmental triggers UV light Smoking Medications – many; include blood Air pollution pressure medications, anti-seizure Trace elements – uranium, lead, medications, antibiotics, interferons Diet mercury, cadmium, nickel, gold, copper, zinc, selenium Oestrogens and hormone Pesticides replacement Bacterial and viral infections Silica (especially Epstein-Barr Virus) Solvents Localised tissue damage, Localised inflammation, causing leading to release of autoantigens up-regulation of co-stimulatory proteins on antigen-presenting cells APCs presenting autoantigens and expressing co-stimulatory molecules Auto-reactive B and T cell activation Autoantibody production SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Cyclic exacerbation of disease Liu & Davidson 2012. Nature Medicine 18:871 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Innate immune system activation All components of the innate immune system are part of the inflammatory response Decreased clearance Increased tissue Decreased anti- Pro-inflammation Infiltrating tissue Reduced number 1)Decreased clearance Pro- Inflammatory of apoptotic cells damage; Increased inflammatory heme by IFNα lesion and of ILC2 leading of apoptotic cells → inflammation responses → prolonging proinflammatory oxygenase-1 (HO-1) promoting tissue to increased prolonging exposure exposure of potential cytokines damage proinflammation of potential auto- auto-antigens production cytokines antigens 2) Decreased elimination of autoreactive B cells SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Cyclic exacerbation of disease Liu & Davidson 2012. Nature Medicine 18:871 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Immune complexes Wang, X., Wang, B. & Wen, Y. npj Vaccines 4, 2 (2019). SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Lupus nephritis: Deposition of immune complexes in kidneys glomeruli Immunofluorescence (IF) staining in lupus nephritis. (A-F) IF findings from a lupus patient with lupus nephritis with different types of immune complex deposition. Parikh SV et al, Am J Kidney Dis. 2020 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Cyclic exacerbation of disease Liu & Davidson 2012. Nature Medicine 18:871 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Clinical disease onset Irreversible tissue damage Rash Renal failure Nephritis Atherosclerosis Arthritis Pulmonary fibrosis Leukopenia Stroke CNS inflammation Carditis Damage from medications: Clotting steroids CURRENT TREATMENT FOR AUTOIMMUNE DISEASES B cell-targeting therapies Examples of B cell-mediated pathogeneses in autoimmune diseases X Lee D.S.W. et al, Nature Review Drug Discovery, 2020 CURRENT TREATMENT FOR AUTOIMMUNE DISEASES B cell-targeting therapies Target: B cell surface markers (CD20, CD19) Drug: anti-CD20 mAb, anti-CD19 mAb B cell depletion ADCC: antibody-dependent cell-mediated cytotoxicity RTX: rituximab (anti-CD20 mAb) CDC: complement-dependent cytotoxicity Guran H. A. et al, Int Immunopharmacol, 2009 Bour-Jordan H. and Bluestone J.A., J Clin Invest. 2007 CURRENT TREATMENT FOR AUTOIMMUNE DISEASES B cell-targeting therapies Target: B cell surface markers (CD20, CD19) Drug: anti-CD20 mAb, anti-CD19 mAb Monoclonal antibodies targeting B cell surface markers approved for autoimmune diseases Rituximab Anti-CD20 mAb Rheumatoid arthritis Ocrelizumab Anti-CD20 mAb Multiple sclerosis Ofatumumab Ineblizumab Anti-CD19 mAb Neuromyelitis optica spectrum disorders CURRENT TREATMENT FOR AUTOIMMUNE DISEASES B cell-targeting therapies Target: B cell survival factor (BAFF) Drug: anti-BAFF mAb BAFF can signal through Belimumab BAFFR, BCMA and TACI human IgG1 recombinant Import for B cell survival monoclonal antibody anti-BAFF throughout B cell (blocking BAFF-mediated signaling) differentiation stages First biologic approved for SLE including immature B cells, (2011) activated B cells, germinal Moderate efficacy center B cells, plasmablasts, plasma cells, memory B cells https://www.biomol.com/ Vana D.R. Biomedical Research, 2018 TREATMENT UNDER INVESTIGATION FOR AUTOIMMUNE DISEASES Some examples of drugs under investigation (in clinical trials) Cytokine-targeting therapies B cell-targeting therapies Kinase-targeting therapies Target IL23 Target B cell survival factor Target BTK (Bruton's tyrosine Mirikizumab (BAFF) kinase) Drug Drug Ianalumab Drug Evobrutinib, Tolebrutinib, Structure AntiI-p19 mAb Fenebrutinib, Rilzabrutinib Structure Anti-BAFF receptor Diseases under Psoriasis, mAb Structure Small molecule BTK investigation ulcerative colitis, inhibitors Crohn’s disease Diseases under SLE, investigation primary Sjögren Diseases under Multiple sclerosis, syndrome investigation pemphigus vulgaris, immune thrombocytopenia Seung Min Jung and Wan-Uk Kim, Immune Network, 2022 POTENTIAL TREATMENT FOR AUTOIMMUNE DISEASES Cell therapies Cell type Features Regulatory T (Treg) cells Disadvantage: can switch from immunosuppressive to effector function CAR (chimeric antigenic Targeting all cell expressing the surface target antigen, e.g. receptor)-T cells CD19-expressing B cells CAR-Treg cells More stable than polyclonal Treg cells CAAR (chimeric autoantigen Specific targeting the B cells producing autoantibodies receptors)-T cells CAAR-NK cells Specific to the B cells producing autoantibodies; better safety than T cells Mesenchymal stem/stromal Limitation: heterogeneity in the MSC culture hence lacking cells (MSC) standardization of treatment protocol Tolerogenic dendritic cells Dampening inflammatory conditions by regulating other immune cells. Limitation: heterogeneity in the DC culture hence lacking standardization of clinical protocol Regulatory B (Breg) cells Cytokine-producing B cells (e.g. IL-10); Limited understanding of how to culture and regulate these cells ex vivo Unconventional T cells (e.g., Non-HLA-restricted, potentially can have a broader gdT cells, MR1-restricted mucosal- application among patients. associated invariant T [MAIT] cells, and CD1-restricted T cells) Fugger L. et al, Cell, 2020 POTENTIAL TREATMENT FOR AUTOIMMUNE DISEASES Targeting metabolic pathways Examples of abnormal metabolic pathways and potential targeting strategies Metabolic pathway Targeting strategy Oxidative Metformin phosphorylation (OXPHOS) GAPDH Dimethylfumarate mTOR Rapamycin/sirolimus, Metformin Glucose transporter CG-5 Hexokinase/Glycolysis 2-deoxyglucose (2-DG) Glucose-6-phosphate 6-aminonicotinamide (6-AN) dehydrogenase (G6PD) ROS Vitamin K3 or buthionine sulfoximine (BOS) Fugger L. et al, Cell, 2020 CURRENT AND POTENTIAL TREATMENT FOR SYSTEMIC LUPUS ERYTHEMATOSUS Examples of treatment tested in clinical trials for SLE: Rituximab Not very effective for SLE Bortezomib Induce plasma cell apoptosis Adverse effects Atacicept Bind both BAFF and APRIL Safety issue CD19-targeting CAR- T cells (Mackensen A. et al, Nat Med, 2022) Tested for refractory SLE High efficacy (5 out of 5 patients responded) Long-term remission unknown Parodis I. et al, Front. Med, 2020 POTENTIAL TARGET FOR AUTOIMMUNE DISEASES Autoantibody-secreting plasma cells Autoantibodies secreted by plasma cells that Immune Memory Laboratory are derived from autoreactive B cells are (Prof David Tarlinton) Inhibition among the key effector molecules in autoimmune diseases Possible strategy: depleting plasma cells while redirecting the immune responses towards non-autoreactive Challenges: Understanding key processes and important genes for plasma cell survival Ideally only targeting the pathogenic plasma cells and not the non-pathogenic ones (Bortezomib treatment has adverse effects such as increased risk of infections due to lack of protective antibodies) FUTURE PERSPECTIVES FOR TREATING AUTOIMMUNE DISEASES New diagnostics Preventive medicine Conventional tests Identify risk factors and groups Antinuclear Antibody Test Polygenic risk scores Autoantibody Test lifestyle and environmental factors Complete Blood Count (CBC) Longitudinal monitoring of the immune Comprehensive Metabolic Panel status of high-risk patients C-reactive Protein (CRP) Test High-throughput analysis of integrated Erythrocyte Sedimentation Rate (ESR) datasets, combining genetic, Urinalysis biochemical, epigenetic, RNA, as well proteomics data New diagnostics Genome-wide screening for genes related to certain autoimmune diseases Detailed disease stratifying Personalised medicine High level data machinery harnessing information that can be collected on a per-patient basis allowing each patient and each disease to be considered independently. Question Set 1 ~15 Minutes 1a. Draw a melanocyte including its specialised morphological feature(s), and where it is located in the layers of the skin. 1b. Why is fair skin a risk factor for the development of skin cancer including melanoma? Explain your answer using your illustration from Q1a (above). 1c. Briefly describe each stage (0-4) of melanoma classification. https://www.aimatmelanoma.org/stages-of-melanoma/ 1d. Consider how each stage of melanoma tumorigenesis relates to the multi- step process of cancer. Allocate each stage (0-4) of melanoma to either initiation, promotion or progression of carcinogenesis. At which stage of melanoma classification do you predict vascular endothelial growth factor (VEGF) expression will be increased – explain your reasoning? Genetic changes drive cell transformation Transformation: Process (transition) from normal cell to cancer cell. Epigenetic and genetic changes lead to: - Loss of tumour suppressor genes expression eg. p53 - Expression of oncogenes eg. mutant EGFR Phenotypic differences between normal and cancer cells Normal Cancer Breast duct Healthy cell Cancer cell Cancer development Initiation: Genetic alteration: spontaneous or induced by a carcinogenic agent. Dysregulation of signaling pathways associated with cellular proliferation, survival & differentiation. Promotion: Epigenetic changes: may be lengthy, during which preneoplastic cells accumulate. May be altered by chemopreventive agents and affect growth rates. Progression: Further genetic changes associated with acquisition of invasive & metastatic potential. Cancer is a multi-step process INVASION Cancer cells require/acquire critical traits to undergo cell transformation to complete the multiple steps essential for development of metastatic cancer METASTASIS The Hallmarks of Cancer Cells Normal cells transform to a neoplastic state, acquiring successive hallmark capabilities and DNA mutations (typically 2-7) Eg. via growth factors Eg. via p53 loss Eg. Via E-cadherin and/or p53 loss Eg. via VEGF Eg. via MMPs and/or loss of E-cadherin Eg. via telomerase Positive and negative regulators of proliferation In normal healthy cells: Heterotypic growth factor and/or integrin signaling promotes proliferation. Cell-cell contact via E-cadherin inhibits proliferation (contact-inhibition). Signal EGF eg.Growth factor, Extracellular matrix ECM Cell Membrane a b E-cadherin Receptor eg.Growth factor receptor, Integrin EGFR Cell Membrane Relay Molecules: Cell-to-cell contact signaling proteins inhibition Pro-proliferation eg. kinase signalling Output: Anti-proliferation effectors signalling Pro-proliferation signalling Pro-proliferative & anti-proliferative pathways are critical for controlling individual cell proliferation & maintaining tissue homeostasis Hallmark 1: Sustained proliferative signalling Cancer cells acquire pro-proliferative signalling 1. Acquire ability to synthesise growth factors - Positive feedback loop (autocrine stimulation) eg. PDGF production by glioblastoma cells PDGF PDGF receptor 2. Overexpression of Growth factor receptors Normal cell Breast cancer cell - Renders cancer cell hyper-responsive to pro-proliferation signals eg. HER2 in breast cancer Hallmark 1: Sustained proliferative signalling 3. Mutation/truncation of Growth factor receptors - Ligand-independent activation of downstream signalling (relay molecules/ effectors) eg. EGFR mutation in lung cancer EGF signalling in lung cancer is altered via multiple mechanisms to EGFR overexpression EGF ligand Mutant EGFR promote proliferation overexpression expression Hallmark 2: Evading growth suppressors Cancer cells are resistant to anti-proliferative signals via E-cadherin loss E-cadherin is a transmembrane protein which connects epithelial cells at adherens junctions: - Suppresses proliferation via cell-to-cell contact inhibition - Tumour suppressor gene E-cadherin Cell Membrane (1) Cell Membrane (2) E-cadherin In cancer, the expression/activity of E-cadherin is lost/ reduced. Hallmark 3: Resisting cell death Cancer cells avoid cell death (apoptosis) via loss of p53 Cell death is required for development and maintaining healthy tissue. During overwhelming cell stress, such as DNA mutations, p53 expression increases to allow Bax/Bak oligomerisation and cytochrome C release leading to intrinsic cell death (apoptosis) P53 is the most commonly mutated tumour suppressor gene in cancer Loss of p53 expression/ function in cancer prevents p53-induced apoptosis. P53/Bcl-XL/Bax, Bak Hallmark 4: Inducing angiogenesis Cancer cells induce angiogenesis Solid tumours (>1-2 mm) require angiogenesis for waste removal and nutrients/oxygen supply. Tumours secrete pro-angiogenic factors (vascular endothelial growth factor, VEGF; fibroblast growth factor, FGF) which bind cognate receptors on endothelial cells to promote angiogenesis. Oncogene expression in cancer cells and/or tumour hypoxia promotes the secretion of angiogenesis factors. Hallmark 5: Enabling replicative immortality Cancer cells display replicative immortality: avoidance of senescence Telomeres are regions of repetitive nucleotides that protects the chromosome end from deterioration or fusion with neighbouring chromosomes. As cells divide, the telomeres shorten. When telomeres shorten to a critical length, the cell undergoes “replicative senescence” (cell stops dividing) Cancer cells often overexpress the enzyme telomerase, which re-generates the telomeres, rendering the cells resistant to replicative senescence Hallmark 6: Activation of invasion & metastasis Cancer cells degrade and invade surrounding tissue: promotes tumour spread A multi-step process involving local invasion, intravasation, extravasation & growth at distant tissue site. Activation of EMT (epithelial to mesenchyme transition) which includes downregulation of E-cadherin. Increased expression/activation of proteins that promote cell invasion eg. matrix metalloproteases (MMP) & Rac Epithelial Mesenchymal Cancer is a multi-step process ALL cancer cells acquire hallmark capabilities Order is variable across the cancer spectrum. One genetic lesion may confer several hallmarks simultaneously eg. p53 or E-cadherin loss, OR Hallmark may require two or more distinct genetic changes. Thus, the number of genetic events for the transformation of a normal cell to cancer cell is variable, 2 to 7 or more... Cancer is a multi-step process Colon cancer arises through different histological stages representing different genetic and epigenetic alterations. Initiation Promotion Expansion/progression Skin Cancer Most common of all cancers in Australia Australia has the highest incidence of skin cancer world-wide at 2-3 times the rate of the U.S.A, Canada and U.K Skin cancer represents ~80% of all cancer diagnosis in Australia 1 in 2 Australians will get skin cancer in their lifetime 80% of skin cancer deaths due to melanoma Basal Cell Carcinoma Squamous Cell Carcinoma Malignant Melanoma BCC SCC MM Basal cells - basal cell carcinoma (BCC) ~80% Squamous cells - squamous cell carcinoma (SCC) ~15% Melanocytes - melanoma (MM malignant melanoma) ~5% INVASIVE POTENTIAL Organisation of skin Skin is the largest organ in the body - Plays many critical roles Basal lamina BCC SCC MM Risk factors for skin cancer Skin cancer divided into: - melanoma and UVB (280-320nm)UVA (320-400nm) - non-melanoma skin cancer which includes SCC and BCC. Common risk factors: * Environment * Immunosuppression * Behaviour * Genetics/family history/complexion/inherited cancer syndrome * Virus * Skin condition Main risk factor for skin cancer is UV radiation UV radiation is the main aetiological factor for skin cancer UV is linked to ~86% of MM, ~70% of BCC and ~60% of SCC However, exposure period, intensity and timing is distinct: - BCC associated with intermittent UV exposure & childhood sunburn - SCC arise from actinic keratosis (precursor skin lesions) & is related to chronic/ cumulative UV exposure & older age - Melanoma: intermittent sun exposure/ severe burning & blistering Major risk factor: Number of moles or dysplastic nevi UVB radiation induces cyclobutane pyrimidine dimers (DNA lesions) UV-B radiation is absorbed by DNA that may lead to DNA lesions including cyclobutane pyrimidine dimers (CPD). Purines Pyrimidines UV light induces pyrimidine dimers: Adenosine (A): Thymine (T) (A:T) - UV light is absorbed by a double bond in C & T bases Guanine (G): Cytosine (C)(G:C) - It physically breaks the double bond -Open bond allows C or T to react to an adjacent base and form a covalent bond When exposed to UV – Adjacent How many CPDs form in Thymines 1 hr PER cell??? UVB Cyclobutane pyrimidine dimers (CPD) UVB radiation induces 6,4 photoproducts (DNA lesions) 6,4 photoproducts occur 3 x less frequently than CPD 5’ base 5’ base 3’ base 3’ base * A covalent bond forms between: C6 of the 5'-base and C4 of the 3'-base (involves the transfer of the hydroxyl group at C4 of the 3'-base) Mechanisms for dealing with UV-induced DNA lesions DNA/nucleotide excision repair (NER) enzymes such as UVrA-D recognises and removes UV-light induced damaged DNA such as CPD. CPD or 6,4 photoproducts induce “kink” in DNA UvrA,B,C endonuclease complex (endonuclease activity) UvrD endonuclease (helicase activity) Typically 11-13 nucleotides Hallmark of UVB-induced DNA damage is a CC-TT mutation DNA exposed to UV light 1 CC dimers are most mutagenic as “translesion polymerases” are biased toward “AA” 2 TC A A TC When the DNA strand (1) with the lesion (CBD) is copied (2), the adjacent cytosines are produced as “AA”,causing them to be paired with thymine (3) resulting in a CC to TT mutation 3 Nucleotide changes in DNA may result in an altered codon. A single amino acid change can have a significant impact on the function/expression/activity of a protein Model for the initiation, promotion and progression of SCC SCC is present in other tissues including lung and colon where it poses a major health risk (compared to skin) COX-2 inhibitors? - Benefits unclear - Conflicting data - Tumours shrink in some studies but not others - Can be associated with: ~ 5 x increase in risk of heart attack and high blood pressure Melanoma Melanoma originates from melanocytes, found in the basal layer of the epidermis and also in the eye. Only known environmental risk factor is exposure to ultraviolet (UV) light. Strong association between childhood blistering sunburn (>3 episodes) & development of melanocytic neoplasia later in life Family history, fair skin and mole number (>20 moles) also important. Melanoma accounts for only ~5% of all skin cancers but is responsible for 80% of deaths from skin cancer; Previously, ~14% of metastatic melanoma patients survive for five years. Melanin levels predict skin color The role of melanin in skin cancer Melanin plays a protective role against UV-induced DNA damage via absorption of UV light Melanocytes secrete melanin vesicles, which surrounding take up Melanin is transported within cells via melanosomes/ granules (small vesicles) Melanosomes accumulate over the nucleus to form a “melanin cap” Melanin absorbs UV light, protecting the nuclear DNA from damage. Studies in mice & humans reveal loss of melanin significantly increases risk of skin cancer including melanoma eg. Albinism & Vitiligo: far greater risk of developing melanoma Albinism Vitiligo B-Raf/MAPK signalling in Melanoma MAPK pathway: major pro-proliferative signalling pathway in melanocytes Upon receptor activation Grb2 and SOS (son of sevenless) are recruited SOS is a Guanine nucleotide exchange factor that activates Ras Ras-GTP binds to and activates B-Raf B-Raf is a serine/threonine kinase that activates MEK Eg. EGFR GTP p p Promotes proliferation, Inhibits apoptosis B-Raf/MAPK signalling in Melanoma ~60% of melanomas contain a BRAF mutation 90% OF BRAF mutations are a single amino acid substitution (Valine to glutamic acid at aa 600; V600E) resulting in 500x increase in BRAF activity, and constitutive activation of MAPK signalling. Mechanism of action Mechanism of action Wildtype B-RAF B-RAF(V600E) acts forms a homodimer as a monomer and binds to independently of Ras-GTP for Ras-GTP activation Ras Ras -GTP -GTP Ras-BD Ras-BD Kinase Kinase domain domain Tissue homeostasis Malignant melanoma Therapeutic approaches targeting B-RAF/ MAPK signalling in Melanoma Previous standard therapies for melanoma patients such as surgery, immunotherapy with IL-2 and conventional cytotoxic chemotherapy: Side effects & result in a poor overall response (only ~5% respond). B-RAF is a target for melanoma treatment: rational design of potent selective B-RAF inhibitors. Vemurafenib/PLX4032/ Plexxikon is a reversible, ATP-competitive inhibitor of the kinase domain of BRAF, particularly the B-Raf V600E Vemurafenib blocks MAPK signalling and induces cell cycle arrest and apoptosis of melanocytes Vemurafenib treatment resulted in a complete or partial response in 81% of melanoma patients. However, within a few months after Vemurafenib treatment the patients relapsed & melanoma re-emerged. Case Studies: Vemurafenib treatment Individuals with B-RAF V600E malignant melanoma PET SCANS 2 weeks after 6 months after 15 weeks after 23 weeks after Before Vemurafenib Vemurafenib Before Vemurafenib Vemurafenib treatment treatment treatment treatment treatment treatment Finn et al (2012). BMC Medicine 10:23 Wagle et al (2011). J Clin Oncol 29:3085 http://www.biomedcentral.com/1741-7015/10/23 What are the potential mechanisms underlying the development of Vemurafenib resistance? Resistance to Vemurafenib in Melanoma Sequencing studies of resistant tumours identified further genetic events Resistance mediated by aberrant splicing (truncation) of the B-RAF(V600E) mutant via deletion of exons, such as exons 2-8: (B-RAF V600E  Ex). Eg. Deletion of exons 4-8 yields p61 B-RAF(V600E) P61 B-RAF(V600E) lacks Ras binding domain and acts as a constitutively active Ras-independent homodimer. Homodimer Monomer More homodimers Highly active & bind strongly to MEK B-RAF and resistance to Vemurafenib B-RAF(V600E): B-RAF V600E  Ex: Monomer Mostly homodimers. Lavoie et al (2011). Nature 480: 329–330 Vemurafenib binds & inhibits Highly active. Vido et al (2018). Cell Reports 25: 1501-1510 downstream MEK activation Vemurafenib binds but cannot inhibit downstream MEK activation FIGURE LEGEND/STUDY NOTE FOR PREVIOUS SLIDE Figure legend from previous slide: a. In normal cells, signal-activated RAS recruits B-RAF to the cell membrane and activates its kinase domain through dimerization. Active B-RAF, in turn, triggers MEK and ERK protein kinases, through phosphorylation (P), to promote cell proliferation and survival. b. The mutant molecule B-RAFV600E constitutively sends signals to MEK and ERK even in the absence of activation by RAS. B-RAFV600E is highly sensitive to inhibition by the anticancer drug vemurafenib. Poulikakos et al. report that B-RAFV600E essentially works as a monomer. c. The authors also show that p61B-RAFV600E — the truncated variant of B-RAFV600E — has an increased propensity to form dimers and that this is associated with resistance to vemurafenib. B-Raf: Vemurafenib, dabrafenib & trametinib Vemurafenib & dabrafenib inhibit BRAF with a V600 mutation (V600E /V600K) Trametinib inhibits MEK (allosteric inhibitor) Rissmann (2015). Br J Clin Pharmacol 80(4): 765 In patients with metastatic melanoma, combined therapies increase progression-free survival compared with single therapy: Dabrafanib: 5.8 months median survival Dabrafanib +Trametinib: 9.4 months Vemurafenib: 7.3 months Vemurafenib +Trametinib: 11.4 months Therapeutic strategies for B-Raf-mutant cancers Colorectal cancer (CRC): common cancer, mortality rate 9.2% second-leading cause of cancer-associated deaths BRAF mutations are found in 10-15% of CRCs BRAF mutations poor prognostic factor Most BRAF mutations in metastatic CRC include V600E Phase 3 Clinical Trial FDA approval Takeda & Sunakawa (2021). Frontiers in Oncology 11 In patients with metastatic colorectal cancers, combined therapies increase median overall survival compared with single therapy. Oncology for B-Raf-mutant cancers: From melanoma to tissue-agnostic therapy Review of sequencing data of 153 554 samples from different tumor types BRAF alterations found in 6% of profiled samples. Prevalence varies across tumor types; highest frequency in thyroid cancer (41%) & melanoma Gouda & Subbiah (2023). ESMO 8 (2) Oncology for B-Raf-mutant cancers: From melanoma to tissue-agnostic therapy Gouda & Subbiah (2023). ESMO 8 (2) Combined therapies show promise across different tumour types Question Set 2 ~10 Minutes 2a. Illustrate what the expression profile of VEGF, MMPs and E-cadherin suggests about the phenotype of the melanoma cells and its surrounding microenvironment? 2b. What is the significance of elevated telomerase activity in the melanomas? How does it impact proliferative potential? 2c. Is the melanoma more likely to be benign or metastatic? Why? BMS3031 Molecular Mechanisms of Disease MELANOMA CASE STUDY You are a research scientist investigating the molecular basis and treatment of Malignant Melanoma [MM]. Of the three types of skin cancers MM is the least prevalent but yet accounts for 80% of skin cancer-related deaths. The majority of MM cases are caused by dominant – activating mutations in the MAP kinase-kinase-kinase protein (MAP3K) B-Raf (Figure 1). However, the genetic basis of a significant proportion of MMs are unknown. Figure 1. Grb2, SOS and Ras activate B-Raf. Activation of receptor tyrosine kinases (RTK) results in recruitment of the adaptor, “growth factor receptor-bound protein 2” (Grb2) and the guanine exchange factor (GEF) “son of sevenless homologue” (Sos). Sos interacts with and activates Ras by exchanging GDP for GTP. Activated Ras-GTP binds to B-Raf to facilitate its activation and subsequent downstream MAPK signaling. 1 BMS3031 Molecular Mechanisms of Disease Your team is involved in a clinical trial for the treatment of patients with malignant melanoma (MM). Initially all of the patients were treated with Vemurafenib [a B- RAF(V600E) mutant inhibitor]. However, half way through the treatment period it was evident that 50% (10/20) of the patients were not responding to Vemurafenib treatment. Therefore, the "non-responding" patients ceased treatment with Vemurafenib, and commenced treatment with a MEK inhibitor. Patients treated with Vemurafenib or the MEK inhibitor showed improved outcomes compared to patients receiving Placebo. All of the 10 patients that were treated with, and responded to a MEK inhibitor were negative for B-RAF mutations. To investigate these melanomas further, your team examines the expression of oncogenic markers and undertakes further molecular analyses which reveals increased expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs), associated with reduced expression of E-cadherin. Moreover, a qPCR-based telomeric repeat amplification (qTRAP) assay revealed elevated telomerase activity. Furthermore, the SOS gene was sequenced in all of the MEK-inhibitor treated patients in an attempt to identify the causative DNA mutation. 5 of the 10 patients had a SOS mutation that changes amino acid Thr266 to Lys [T266K] (Figure 2). In vitro, cell culture expression of the SOS(Thr266K) mutant leads to increased levels of phosphorylated ERK compared to cells expressing wild-type SOS. The remaining 5 patients did not have a mutation in SOS, and the causative mutation was not identified. The 50% of patients (10/20) with a B-RAF mutation expressed the classical B- RAF(V600E) mutant. After 9 months of Vemurafenib treatment, 100% of the B- RAF(V600E) mutant patients developed resistance to Vemurafenib and the MM relapsed. Figure 2. Domain structure of SOS. Sos GEF is a 1333 aa protein with a Dbl Homology (DH) and a Pleckstrin Homology (PH) domain that is essential for its GEF activity and thereby activation of Ras. Red arrow indicates the site of the T266K mutation. 2 Question Set 3 ~10 Minutes 3a. What effect if any do you predict the T266K mutation has on Sos activity and function? 3b. What effect if any do you predict the Sos T266K mutation has on signalling components upstream of Sos? 3c. What effect if any do you predict the Sos T266K mutation has on signalling components downstream of Sos, specifically Ras and MEK1/2? Note: For your answer, draw the MAPK signalling pathway including all of the signalling components and where in the pathway Vemurafenib acts. Question Set 4 ~10 Minutes 4a. Five patients in the trial that did NOT respond to the initial Vemurafenib treatment, but did respond to the subsequent MEK inhibitor, did not have a B- RAF or Sos mutation. Using this information, where in the signalling pathways do you predict there may be a causative mutation? Explain your answer. 4b. In reference to the patients in Q4a, which signalling component(s) would you investigate next for gene mutation(s) as a potential causative candidate(s)? Explain your answer. 4c. What is the likely mechanism of Vemurafenib-resistance in the patients with the BRAF(V600E) mutation? Illustrate how the mechanism of action of the resistance mutant differs to BRAF(V600E) in the context of MAPK signalling. Monoclonal antibody therapy The first mAbs generated were made in mice – great diagnostic benefits but therapeutic benefit was limited due to: * host immune response to mAb * mAb clearance To improve the potential of the therapeutic monoclonal antibody, “Humanisation” of the mAb was undertaken – mid 1990’s Monoclonal antibody therapies Mechanism of action of therapeutic mAbs Indirect- not dependent on the specific antigen recognised by the antibody ADCC: Antibody dependent cellular cytotoxicity. FcγR binding of Fc portion of Ab. NK cells activated via ITAM motif in FcR, cytotoxic granules are released, inducing cell death. CDC or CMC: complement-dependent cytotoxicity or complement ADCP mediated cytotoxicity Activation of complement cascade, cell lysis. Binding of C1q to Fc part of antibody. Cell death occurs via formation of the membrane attack complex (MAC), which consists of complement proteins C5b, ADCP: Antibody dependent cellular phagocytosis. C6, C7, and C8 and various copies of C9, generating pores in the membrane. FcγRs on phagocytic cells eg macrophages, bind to Fc portion of Ab, leading to internalisation and degradation of target tumour cell. Also promotes antigen presentation of antigens from the phagocytosed cell. 9 Mechanism of action of therapeutic mAbs Direct- dependent on the specific antigen recognised by the antibody Antigen–specific antibodies bind their antigen, eg cell surface receptor. Antigen-specific binding can lead to: - Blocking of ligand binding site, preventing receptor signalling - Inhibition of dimerization of receptor, preventing receptor signalling - Blocking of survival, growth signals - Apoptosis of target cell 10 Therapeutic antibodies can be utilised as single agents or delivery mechanisms for other modes of therapies Antibodies can be used as single ‘naked’ agents to directly target cells or conjugated to for eg: Cytokines, drugs, radioisotopes, toxic agents, antigens to enhance therapies Discovery of HER2 amplified in breast cancer  1984 – Weinberg characterizes an oncogenic rat version of HER2/erbB2, calls it ‘neu’  1985 – Genentech team clone human HER2/erbB2  1987 – Demonstration that HER2 amplification in breast cancer occurs in ~ 25% of patients and is associated with more aggressive disease Single copy 2-5 copies 5-20 copies Southern blot analysis of HER2 gene copy number in breast cancer specimens Slamon et al, Science. 1987 235(4785):177-82 14 HER2 receptor: HER2, encoded by the gene ERBB2. Official name erb-b2 receptor tyrosine kinase 2 HER2 is a member of the EGFR family: HER1, HER2, HER3 and HER4 - Her1, 2 and 4 but not HER3 exhibit tyrosine kinase activity (cytoplasmic tail) HER1, 2 & 4 are composed of 3 domains: Extracellular – binds ligand Transmembrane – anchors receptor in cell membrane Intracellular/kinase domain – contains tyrosine kinase domain HER2 does not bind ligands, is activated via hetrodimerisation with HER1/3/4. - HER2 e/c domain adopts a conformation resembling a ligand-activated state which allows hetrodimersation in the absence of ligand HER1, 3 and 4 bind multiple ligands - HER2 can shed its e/c domain rendering It constitutively active -HER2 activates multiple signalling pathways including MAP kinase and PI3-kinase -HER2 promotes cell growth, survival, cell cycle progression, angiogenesis (hallmarks of cancer) HER2 receptor and breast cancer In ~25% of breast cancers HER2 shows: - Amplification (multiple HER2 genes) -Overexpression (many HER2 receptors/proteins) HER2 is a proto-oncogene H E Amplification of HER2 is a significant predictor of both overall survival and time to relapse. - HER2 amplification/overexpression is associated with reduced survival TARGETED THERAPY Slamon et al, Science 1987 Trastuzumab (trade name Herceptin) Just the CDR regions are mouse Binds extracellular domain of HER2 Blocks HER2 heterodimerisation Blocks HER tyrosine kinase activity Blocks e/c domain C-cbl ubiquitin shedding ligase Promotes HER2 degradation G1 arrest ADCC Results of Phase III trials: Trastuzumab/Herceptin increases the clinical benefit of first-line chemotherapy in metastatic breast cancer that overexpresses HER2. - Cell effects: Reduces downstream MAPK/PI3-kinase signalling, Inhibits proliferation and angiogenesis, Arrest in G1 of cell cycle, Promotes apoptosis - HER2 effects: Inhibits HER2 e/c domain shedding, Promotes HER2 degradation, inhibits HER2 heterodimerisation, Inhibits HER2 tyrosine kinase activity - Associated with cardiac dysfunction in ~5% of cases (N Engl J Med 2001; 344:783-92.) 17 Positive HER2 status: a predictive biomarker for patient response 2+ tumours get additional testing for HER2 amplification Not eligible Not eligible eligible eligible IHC Normal 0 Normal 1+ Abnormal 2+ Abnormal 3+ FISH Normal Normal Abnormal, low Abnormal, high amplification amplification IHC Images courtesy of MJ Kornstein, MD, Medical College of Virginia Herceptin resistance -Expression of the truncated p95HER2 (lacks e/c domain to prevent drug binding) - Signalling by other HERs - Hyperactivation of MAPK or PI3-kinase (via mutations in pathway components) - Overcoming resistance: Combination treatments depending on mechanism of resistance. Recent developments in anti-HER2 therapy Pertuzumab (Perjeta) – binds HER2, blocks association with HER3 2012 CLEOPATRA trial – confirmed improved efficacy of pertuzumab+trastuzumab+docetaxel in HER2-positive metastatic breast cancer Initiating T cell activation: role of dendritic cells Found in peripheral tissues Sample environment via cellular extensions Act as sentinals/scouts for any infection Initiating T cell activation: role of dendritic cells “Immature” Infection/TLR activation “Mature” Increased processing Upregulation of costimulatory molecules Secretion of key cytokines Migration to find Tcells in lymph nodes B cell activation Requires: Cross linking of antigen receptors B cell receptor (BCR) Second signals (co- stimulation/cytokines) Differentiation results in formation of memory/plasma cells Location of B cell response Distribution of cells in the lymph node cortex & medulla “Helped” B cells move into germinal centers Evolution of antibody responses Antibody response “matures” by: Isotype (class) switching (IgM IgG IgA IgE) Affinity maturation High affinity IgG concentration Low affinity Antibody IgM/IgG 10-14 days Months/Years Infection (pathogen A) Re-Infection (Pathogen A) The small pox vaccine induced long term “cross- protective” immunity Hammerlund et al, Nat Med 9:1131-1137, 2003 Neutralising Antibody concentration 1x vaccination 2x vaccination 3x vaccination 1 yr Years after vaccination 70 yrs Smallpox vaccination Yellow fever vaccine: The Gold stadard The yellow fever vaccine YF-17D is one of the most successful vaccines ever developed Yellow fever symptoms : Its success is linked to activation of dendritic High fever cells via multiple Toll-like Receptors (TLRs) Bleeding into the skin Liver and kidney stimulating proinflammatory cytokines cytotoxicity Jaundice Yellow fever virus YF-17D vaccine activates multiple TLR signals Trif Myd88 IRF3, IRF7, NFB Approach Nanoparticles made of a biodegradable polymer Synthetic Toll-like receptor ligands Antigens MPL – lipopolysaccharide derived adjuvant (TLR4) R837 – TLR7 ligand R848 – TLR7 and TLR8 ligand Poly-D, L-lactic-co-glycolic acid Ovalbumin Influenza HA Protective antigen Bacillus anthracis (Anthrax) SILDENAFIL: THE LITTLE BLUE PILL  Pfizer discovered Sildenafil in 1989 while looking for a treatment for heart-related chest pain.  Sildenafil acts by blocking phosphodiesterase 5 (PDE5), an enzyme that promotes breakdown of cGMP.  This results in relaxation and dilation of some blood vessels, particularly in the penis and lungs.  While sildenafil improves some markers of disease in people with pulmonary arterial hypertension, it does not appear to affect the risk of death. Event Modifier Prenatal perturbation Nature, timing, duration, Barker (eg. poor nutrition, glucocorticoids, GDM) severity of insult Hypothesis Altered embryonic/ Gender fetal development results in LBW and Altered kidney development Compensatory renal changes predisposition for adult (eg. branching morphogenesis) (eg. gene expression, hypertrophy) disease (CV, metabolic, renal etc.) Postnatal growth Low nephron Low podocyte (eg. lactational environment, endowment endowment “catch up” growth) Compensatory renal changes (eg. glomerular hypertrophy, Brenner asymmetric nephron growth) Hypothesis Lower filtration Glomerular Low nephron number surface area sclerosis Lifestyle predisposes to adult (eg. salt/fat/protein in diet) hypertension and CKD Hypertension CKD/ESKD Overall Summary  The concept that chronic diseases are initiated through processes that occur before birth and/or shortly after birth emerged 30-40 years ago.  The early evidence was circumstantial and the mechanisms unknown.  Development programming of non-communicable diseases is now an established paradigm.  The most commonly used surrogate index of “developmental success” is birth weight – a crude index but still useful and increasingly recorded.  Low birth weight and premature birth are associated with increased risk for many adult chronic diseases, including hypertension and CKD.  Optimization of maternal health and early childhood nutrition can attenuate this programming cycle and reduce the global burden of chronic disease. Early screening and education programmes are warranted.  Non-invasive kidney imaging will be useful

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