Exam 1 - DNA Mutations - PDF

Exam 1 2/17/25 (Monday) 10:00 am - 11:15 am S 105 (in-person in-class in Canvas) Lecture 1-8 Please update your lockdown browser before the exam! Check out with me after submitting the exam before leaving the classroom! 45 multiple choice questions, 2 pts each 1 shor...

Exam 1 2/17/25 (Monday) 10:00 am - 11:15 am S 105 (in-person in-class in Canvas) Lecture 1-8 Please update your lockdown browser before the exam! Check out with me after submitting the exam before leaving the classroom! 45 multiple choice questions, 2 pts each 1 short answered question, 10 pts. Review for exam-1 Cancer terms: Carcinomas: Malignant tumors of the epithelium. Sarcomas: Malignant tumors of the mesenchyme (e.g. bone, muscle) Adenocarcinomas: Malignant tumors of a gland (e.g. breast cancer). Angiogenesis: the formation of new blood vessels Metastasis: The process of cancer cells spreading from a primary site to secondary sites in the body.  Types of DNA mutations the substitution of one purine for another purine the substitution of a purine for a pyrimidine or vice versa © Oxford University Press, 2021 4  DNA mutations Additional mutations identified from recent advances in sequencing technology and imaging: Kataegis (Greek for “thunderstorm”): Small localized areas of hypermutation. Chromothripsis (“chromo” meaning chromosome and “thripsis” meaning breaking into small pieces): A one-off cell crisis that shatters chromosomes and results in tens to hundreds of genomic rearrangements, leading to multiple mutations. kataegis vs chromothripsis  Chromosomal and circular extrachromosomal DNA Tumor cells contain large circular ecDNAs (100 Kb to 1.3 Mb in size) that are found in one in four solid tumors, but rarely in normal cells. ecDNAs can replicate but do not have centromeres, they are not equally segregated to daughter cells upon cell division. This leads to extreme copy number amplification and contributes to tumor cell heterogeneity. ecDNAs may be a source of mutagenesis through the formation of amplified genes, chimeric rearrangements, and reintegration into the linear genome of chromosomes. ecDNA: DNA that does not reside on a chromosome. A distinct type is commonly observed in human cancer cells © Oxfordand isPress, University known2021 to carry oncogene. 7  mechanisms to prevent and repair mutations The human genome is continually exposed to numerous mutagens, both endogenous and exogenous.  Sophisticated DNA repair mechanisms prevent and repair mutations DNA replication is a potential source of genetic instability.  Minimized by exonuclease proofreading activity of replicative DNA polymerases  post-replication surveillance by the mismatch repair (MMR) system. Failure of these mechanisms in tumors is associated with a very high mutation burden---”hypermutation” or “ultramutation”.  mechanisms maintaining genomic stability- mismatch repair The fidelity of DNA duplication is further increased by mismatch repair (MMR) system. MMR corrects replication errors that have escaped editing by polymerases. It includes base–base mismatches, as well as insertions and deletions produced as a result of slippage during the replication of repetitive sequences (microsatellites). insertion deletion For historical reasons, highly repeated sequences in the genome, often carrying 100 or more nucleotides per repeat unit, have been called “satellite” sequences. Far shorter repetitive sequences found in many places in the genome have been named “microsatellite”.  Mismatch repair deficiency (Lynch syndrome) Lynch syndrome: hereditary non-polyposis colon cancer (HNPCC). HNPCC is an autosomal dominant condition that predisposes sufferers to various malignancies including colorectal, ovarian, endometrial, stomach, and prostate cancer. HNPCC is responsible for 2 to 3% of all colon cancer cases. The majority (85-90%) of HNPCC cases result from germ-line mutations in the genes encoding two important mismatch repair proteins, MSH2 and MLH1. MSH6 and PMS2 mutation are also involved in a small number of cases (about 15%). Lecture 2 mismatch repair mechanism Two MMR genes-MSH2 and MLH1-are commonly involved in HNPCC. MSH6 and PMS2 mutation are also involved in a small number of cases. PMS1 may also be involved in a small number of cases. MMR is initiated by the binding of MutS (2 main forms) to the aberrant DNA region. hMutL heterodimers, including hMLH1/hPMS2 and hMHL1/hPMS1, are recruited.  Mismatch repair deficiency (Lynch syndrome) The inability to properly detect and repair sequence mismatches leads to high rates of mutations in genes that have microsatellite repeats nested in their sequences (microsatellite instability) as well as a very high mutation rate overall. Most Lynch syndrome-associated tumors display microsatellite instability, particularly among cases with MSH6 variants.  Polymerase proofreading domain mutations (sporadic ultramutated cancers) Germ-line mutations in the human PolD1 and PolE genes (catalytic subunits of pol-δ and pol-ε) lead to predisposition to colon cancer and other malignancies. Somatic mutations in the proofreading-encoding domains (exonuclease domain) of PolD1 and PolE genes have been observed in endometrial cancer, colon cancer and other tumor types. Tumors carrying these mutations exhibit “ultra-mutated” phenotype, which leads to as many as one million base substitutions in the genomes of certain tumors. In contrast to MMR-deficient tumors, these tumors do not exhibit microsatellite instability. Sporadic tumors with somatic POLE exonuclease domain mutations have the highest burden of mutation of any tumor type.  APOBEC overexpression APOBEC (apolipoprotein B mRNA editing catalytic polypeptide) overexpression: dysregulation of mechanisms designed to promote mutagenesis Normal function of APOBEC: APOBEC is a family of cytidine deaminase playing important roles in pathogen defense by inducing DNA damage of infecting RNA and DNA viruses. APOBECs also serve vital roles in innate immunity: aid antibody diversification by causing somatic hypermutation (C to T mutations) at the antigen-binding sites  APOBEC overexpression C>T mutations. C>G mutations from DNA repair intermediates such as abasic sites APOBEC overexpression (APOBEC3A, APOBEC3B, APOBEC1) is a major contributor to genetic instability in human cancers:  responsible for kataegis  induce cancer.  drive cancer drug resistance (TKI resistance in lung cancer and enzalutamide resistance in prostate cancer) APOBEC mutation signature accounts for the majority of mutations in 10% ER positive breast cancers. Summary of DNA repair pathways Key mutated proteins BRCA1/BRCA2 XP (XP syndrome) ERCC (Cockayne syndrome ) MUTYH (MAP) PARP MSH2, MLH1 (Lynch syndrome) MSH6, PMS2  Comparison of three repair mechanisms Oxidation (8- OGG1 oxoguanine), MUTYH deamination, PARP alkylation MSH2, MLH1 MSH6, PMS2 XP ERCC Targeting PARP in BRCA mutant cancer (synthetic lethal interactions) Mouse ES cells treated with PARP1 inhibitor, KU0058948  Nucleotide excision repair defects (XP) Xeroderma pigmentosum (XP) syndrome In 1874, two Austro-Hungarian physicians, Ferdinand Hebra and Moritz Kaposi, described an unusual syndrome that involved high rates of the development of squamous and basal cell carcinomas of the skin. Affected individuals have extreme sensitivity to UV and infants suffer severe skin burning after only minimal exposure to sunlight. XP individuals have a 2000-fold increased risk of skin cancer before age 20, and about a 100,000-fold increased risk of squamous cell carcinoma of the tip of the tongue. NMSC: nonmelanoma skin cancer XP patient has severe lesions in sun-exposed area  Base excision repair defects (MUTYH) The first step of BER is carried out by a family of DNA damage- specific glycosylases (such as OGG1 and MUTYH). Inherited defects in BER due to mutations in the MUTYH cause a multiple colorectal adenoma syndrome, called MUTYH-associated polyposis (MAP). Patients with MAP have increased lifetime risk of developing colorectal cancer: 43% rising to 100% in the absence of surveillance. These individuals are also prone to development of polyps of the upper gastrointestinal tract and cutaneous tumors. Tumors in MAP patients display a characteristic mutation spectrum with a marked increase in G>T transversion mutations.  Chemotherapy Three main types:  Alkylating agents and platinum-based drugs  Antimetabolites  Organic drugs  Alkylating agents and platinum-based drugs Alkylating agents  nitrogen mustards---cyclophosphamide, ifosfamide, melphalan, chlorambucil;  nitrosoureas---carmustine, loumustine, streptozotocin  alkyl suphonate---busulfan Their principal mechanisms of action is to alkylate the N7 residue of the guanine which can cross-link nucleobases in DNA double-helix strands. This makes the strands unable to uncoil and separate leading ultimately to apoptotic cell death. Platinum-based agents, including cisplatin, carboplatin, oxaliplatin  form covalent bonds via the platinum atom.  also bind N7 guanine and can cause intra- and interstrand DNA crosslinks, inhibiting DNA synthesis and inducing programmed cell death.  Antimetabolites Antimetabolites are agents that resemble an endogenous metabolite and block a metabolic pathway.  Purine analogues (mercaptopurine, thioguanine, fludarabine, pentostatin, and cladribine): mimic metabolic purines, inhibit DNA synthesis  Pyrimidine analogues (5-fluorouracil, gemcitabine, floxuridine, cytosine arabinoside): mimic metabolic pyrimidines, inhibit DNA and RNA synthesis  Antifolate analogues (methotrexate, pemetrexed): drugs impair the function of folic acids. Methotrexate: folic acid analogue, binds and inhibits dihydrofolate reductase (DHFR), prevents tetrahydrofolate formation which is essential for purine and pyrimidine synthesis and protein production (Ser and Met synthesis) BCHS3305 Action of antimetabolites fluorodeoxyuridylate (F- dUMP) and methotrexate Drugs inhibit DNA synthesis Thymidylate synthase uses N5N10 methylene-tetrahydrofolate as a methyl donor and catalyzes the methylation of dUMP to form dTMP.  Organic drugs  Anthracyclines (doxorubicin, daunomycin, epirubicin, daunorubicin): antitumor antibiotics derived from the Striptomyces bacteria.  Three mechanism of actions:  Inhibits DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand, preventing the replication of rapidly-growing cancer cells.  Inhibits topoisomerase II enzyme, preventing the relaxing of super-coiled DNA and blocking DNA transcription and replication.  Creates iron-mediated free oxygen radicals that damage the DNA and lipid domain of cell membranes. Cardiac damage is its most severe side effect, but new compounds (e.g. ICRF-187) that can block the cardiac toxicity are being investigated. These drugs are primarily used to treat solid tumors (e.g. of the breast or lung).  Organic drugs  Vinca alkaloids (vincristine, vinorelbine, vinblastine, vindesine): antimitotic and antimicrotubule agents, originally derived from the Periwinkle plant Catharanthus roseus.  Principle mechanisms of cytotoxicity:  Interaction with tubulin and disruption of microtubule function, particularly of microtubules comprising the mitotic spindle apparatus, leading to metaphase arrest.  Organic drugs  Taxanes (docetaxel, paclitaxel, nab-paclitaxel): diterpenes produced by the plants of the genus Txus (yews).  Principle mechanism of action:  Disruption of microtubule function.  Microtubules are essential for cell division. Taxanes stabilize GDP-bound tubulin in the microtubule, inhibiting the process of cell division – a ‘frozen mitosis”.  Taxanes are mitotic inhibitors  In contrast to the taxanes, the vinca alkaloids destroy mitotic spindles.  Both taxanes and vinca alkaloids are together named spindle poisons. Taxanes and Vinca alkaloids inhibit mitotic spindle  Mechanisms of drug resistance Anticancer drugs impose a strong force for the selection of cells that can acquire drug resistance. increasing the efflux of the drug decreasing the intake of the drug increasing the number of target molecules within the cell altering drug metabolism Altering DNA repair processes Reference© Oxford reading: Universityhttps://www.nature.com/articles/nrc706 Press, 2021 29  Increasing the drug efflux After selection for resistance to a single drug, cells might also show cross-resistance to other structurally and mechanistically unrelated drugs — a phenomenon known as MULTIDRUG RESISTANCE. — generally results from expression of ATP-dependent efflux pumps with broad drug specificity. These pumps belong to a family of ATP-binding cassette (ABC) transporters, including MDR1 (multi-drug resistance gene, P-glycoprotein (P-gp), ABCB1), MRP1 (ABCC1) and ABCG2. MDR1, normally a chloride ion efflux pump, can bind a variety of chemotherapeutic drugs. Upon binding, ATP is hydrolyzed and causes a conformational change of MDR1. The drug is released extracellularly.  Affected drugs: VINCA ALKALOIDS (vinblastine , vincristine), the ANTHRACYCLINES (dox orubicin and daunorubicin), the RNA transcription inhibitor actinomycin-D; the microtubule-stabilizing drug paclitaxel.  Super-enhancer Super-enhancer: Large clusters of multiple distinct TFs (each binding to its own enhancer sequence) assemble together with general TFs to drive high-level transcription of nearby non-housekeeping genes. During tumorigenesis, tumor cells acquire specific SEs to promote oncogene expression, which mediates the dysregulation of signaling pathways Additional reading: https://www.nature.com/articles/s12276-020-0428-  Acetylation of histones neutralizes the positive charge on lysine residues and relaxes chromatin folding Histone acetylation correlates with enhanced transcriptional elongation by RNA polymerase II. Histone deacetylation restores a positive charge to lysine, stabilizes chromatin compaction and higher-level packaging, limits the accessibility of TFs and results in the transcription repression. Residues in all four of the core histones can be acetylated but transcriptional activation is particularly associated with acetylation of H3K9 and H3K14, H3K27. Many HATs can also acetylate other non-histone proteins such as transcription factors p53 and E2F. KAT: Lysine acetyltransferase  DNA methylation DNA methylation is the addition of a methyl group to position 5 of cytosine. Only 3–4% of all cytosines in DNA are methylated. Methylation only occurs at cytosine nucleotides which are situated 5ʹ to guanine nucleotides (CpGs). DNA methylation and CpG island CpG clusters, called CpG islands, are located in the promoter region of 50% of human genes. In general, the CpG islands found in gene promoter regions are not methylated in normal tissues, and transcription may occur. Methylated cytosines are found mainly in repetitive sequences and in the CpG islands found in the promoter region of repressed genes such as X-chromosome inactivated genes, imprinted genes, and some tissue-specific genes. CpG islands of tumor suppressor genes are commonly hypermethylated in many different cancer cells, resulting in epigenetic gene silencing.  DNA methyltransferase Three methyltransferases are known: DNMT1, DNMT3a, and DNMT3b. DNMT1 is involved in the conversion of hemi-methylated DNA to fully methylated DNA during replication. This mechanism allows methylation patterns to be inheritable; if only one strand remained methylated, the signal would be lost in half of its daughter cells after replication. The other two methyltransferases are mainly involved in de novo methyltransferase activity (methylation of new sites).  Intratumor epigenetic alterations  aberrant DNA methylation Abundant TSGs are under hypermethylation e.g. RASSF10 in kidney cancer, SIX3 in glioblastoma, CDKN2A and PTEN in melanoma Additional genes involved in multiple pivotal cellular functions also present hypermethylation. e.g. prostate cancer (PC) have demonstrated that the heavily methylated situation occurred on the promoters of glutathione S-transferase pi (GSTP1) and other genes such as CDKN2A, TIMPS, and DAPK, which participate in cell cycle, cell metastasis, apoptosis etc. Hypomethylation of oncogenes e.g. LY6K in glioblastoma, SLC34A2 in papillary thyroid carcinoma, RBBP6 in colorectal cancer  Bromodomain and extra terminal inhibitors (BETIs)  Bromodomains are ‘readers’ of acetylated lysine residues and found in over 40 proteins in human including HATs and HDACs. The BET (bromodomain and extra-terminal domain) family proteins, consisting of BRD2, BRD3, BRD4, and testis-specific BRDT. They are characterized by two tandem bromodomains (BDs) that bind to lysine-acetylated histones and transcription factors, recruit transcription factors and coactivators to target gene sites, and activate RNA polymerase II machinery for transcriptional elongation.  BRD4 and BETI BRD4 is the most characterized BETs that is assembled on both hyper-acetylated gene promoters and “super-enhancers” to promote RNA-pol II-mediated transcriptional initiation and elongation Several oncogenes have been described as the effectors of BRD4, including c-Myc, FOSL1, RUNX2, BCL-2, and c-KIT The efficacy of BETIs is based on the disruption of BETs- acetylated histones interaction. Several BETIs have encouraging clinical outcomes with tolerable toxicity and potent efficacy, including thienodiazepine JQ1, I-BET762 (GSK525762), I-BET151 (GSK1210151A), GS-5829, CPI-0610, TEN- 010, OTX-015, and ZEN003694.  BETI: JQ1 JQ1 has been reported to block the interactions of BRD2/3/4 and acetylated histones selectively One of the target genes c-Myc is subject to downregulation by JQ1 in many different cancers It exhibits a drastically anti-tumor activity even in castration- resistant cells by disrupting BRD4-mediated androgen receptor (AR) recruitment and transactivation. JQ1 also increased cytotoxic T-cell response by increasing PD-L1 expression Histone lysine methylation and KMTs  Histone methyltransferases inhibitors EZH2 is a histone methyltransferase and is responsible for the catalytic activity of PRC2 (Polycomb Group). EZH2 overexpression has been detected in a wide range of malignancies. EZH2/PRC2 methylates H3K27 and leads to transcriptional silence of target genes in multiple subtypes of cancers, including ovarian cancer, breast cancer, PC, T-cell ALL and non-Hodgkin lymphoma  Histone methyltransferases inhibitors Small molecule EZH2 inhibitors, such as EPZ-6438 (tazemetostat), GSK2816126, and CPI-1205, have been evaluated in clinical trials and showed antineoplastic effects in both hematologic malignancies and various solid tumors. EPZ-6438 is an orally bioavailable EZH2 inhibitor by competing with SAM, which is a cofactor of EZH2 Year 2020 Tumor suppressors Non-coding RNA  miRNA (micro RNA) small, non-protein-coding RNAs (18–25 nucleotides in length) Each miRNA may be able to repress hundreds of gene targets post-transcriptionally. Steps: 1. transcribed by RNA polymerase II from intergenic regions or from regions that code for introns 2. the primary transcript is processed by ribonucleases Drosha and DGCR8 in the nucleus-----pre- miRNAs, hairpin-shaped intermediates of 70–100 nucleotides.  circRNA (Circular RNA) Generated from backsplicing (a spliceosome utilizes 3′ splice site that is upstream of the selected 5′ splice site) form covalently closed continuous loops long half-lives due to the lack of free 3′ or 5′ ends lncRNA function  lncRNA examples  HOTAIR (HOX antisense intergenic RNA) Deregulation of HOTAIR is associated with cancer progression in 26 tumor types and overexpression is Inhibit miR-7 predictive of metastasis in breast cancer. leading to gene overexpression guides chromatin-modifying factors to specific sites to facilitate epigenetic regulation. function as a sponge by binding to, and inhibiting, the repressive function of specific miRNAs SNAIL/HOTAIR/EZH2 complex, inhibit Promote expression of epithelial prometastatic genes activity  Telomere and Telomerase Telomeres are composed of several thousand repeats of the sequence TTAGGG.  Telomerase Telomerase, a ribonucleoprotein containing human telomerase reverse transcriptase (TERT) (enzyme that synthesize DNA from RNA) and a human telomerase RNA (hTR) maintains telomere length in certain cell types such as stem cells. Expression of telomerase is tightly controlled in differentiated cells. Most cancer cells reactivate telomerase to escape of the fate of cellular aging.  The cell cycle is unidirectional The main mechanism for unidirectionality: the strictly ordered periodic expression of cyclins followed by their abrupt destruction. CDK activity is low in early G1 phase due to the absence of cyclins. Cyclin D is the first cyclin to be synthesized and, in complex with CDK4 and CDK6, drives progression through G1. Cyclin D regulates expression of the cyclin E gene--- important for the G1 to S phase transition. Cyclin D becomes degraded during the G1 to S transition, preventing erroneously triggering a new round of cell cycle entry during later stages of the cell cycle.  The cell cycle is unidirectional Cyclin A–CDK2 is important for S phase progression, whereas the cyclin A–CDK1 and cyclin B–CDK1 complexes direct G2 and the transition from G2 to M phase. Cyclin A levels start declining during G2, whereas the rapid degradation of cyclin B upon mitotic entry is necessary for exit from mitosis and subsequent cytokinesis. Cyclin levels in the daughter cells are low, resetting the cell to the state of low CDK activity that characterizes G1.  Mechanisms of CDK regulation CDK needs to be phosphorylated by CAK (CDK activating kinase) and to associate with a cyclin to become fully active. CDK activity is negatively regulated by CKIs, which physically associate with CDK to inhibit its activity CIP/KIP/CDKN1-family CKIs, such as p21Cip, p27Kip associate with the cyclin–CDK1 holoenzyme. members of the INK4/CDKN2 family, such as p16, form a complex with CDK in the absence of a cyclin. CDK activity can also be inhibited by phosphorylation at Thr14 and Tyr15 by WEE1 kinase. These phosphorylation are removed by CDC25 phosphatase, which is required for full CDK activity. Two steps are required for CDKs to become active: dephosphorylation of the inhibitory phosphate groups by CDC25 phosphatases, and phosphorylation of a central threonine residue by CAK  CKIs  INK4 family: o p16INK4a, p15INK4b, p18INK4c and p19INK4d o specific for CDK4 and CDK6 o In response to anti-proliferative signals, INK4 proteins are transcribed and bind CDK4 and CDK6 causing a conformational change which reduces their affinity for D-type cyclins  The Cip/Kip family: o p21Cip1/Waf, p27Kip1 and p57Kip2. o bind CDK4/6-cyclin D and CDK-cyclin A/B/E complexes CKIs  Regulation of CDK activity by phosphorylation Activation of CDK requires phosphorylation of a C-terminal threonine by the CAK. Phosphorylation of N-terminal threonine and tyrosine residues near the ATP binding pocket is inhibitory (prevents ATP binding)—by Wee1 and Myt1 Phosphorylation of CDK1-2 contributes to the timing of their activation during a normal cell cycle inhibition activation activation The CDK4/6 enzymes appear to be subject to this inhibitory phosphorylation only when cells incur DNA damage.  Neuregulin EGF receptor family members: EGFR (ERBB1 or HER1) ERBB2 (HER2) ERBB3 (HER3) ERBB4 (HER4) HER2 does not bind to a known ligand but acts as a co-receptor for the other members of the family HER3 has only weak kinase activity. ERBB: erythroblastic leukemia viral oncogene homologue HER: human epidermal growth factor receptor  Grb2 Grb2: Growth factor receptor bound protein-2 A 25 KDa adaptor protein consists of a Src homology 2 (SH2) domain surrounded by two Src homology 3 (SH3) domains. Acts as an intermediate switch between cell surface activated receptors and downstream targets. Grb2-SH2 binds phosphorylated pYXNX motifs at RTK, FAK, IRS-1, SHP-2 Grb2-SH3 links to downstream effector: son of sevenless (SOS), a guanine nucleotide exchange factor (GEF) https://juniperpublishers.com/c toij/pdf/CTOIJ.MS.ID.555618.pdf  SH2 and SH3 domain SH2 domains (approximately 100 amino acids long) : recognize and bind to distinct amino acid sequences (1–6 residues) C-terminal to phosphorylated tyrosine residues SH3 domains (approximately 50 amino acids long): recognize and bind to proline and hydrophobic amino acid residues on partner proteins. Both SH2 and SH3 domains are frequently found in the same protein, e.g. GRB2, SRC, ABL, and PI3K. GRB2 interact with the SH3 domains of exchange protein SOS (son of sevenless), translocating it to the membrane. It is this translocation to the membrane which enables the activation of the pivotal membrane-bound intracellular transducer RAS.  RAS activation Three members of the RAS family N-, H-, and K-RAS 21KDa GTP-binding proteins Inactive: GDP-bound active: GTP-bound GEFs (guanine nucleotide exchange factors), such as SOS, catalyze the release of GDP from the guanine nucleotide binding pocket on RAS GTP is tenfold more abundant in the cytoplasm, it then binds to RAS GTP binding causes a conformational change and results in RAS activation, allowing interaction with downstream signal transducers GAPs (GTPase activating proteins) accelerate the hydrolysis of GTP to GDP to terminate the signal.  The structure of RAS protein G12, G13, Q61 mutations Reduce or eliminate GTPase activity (compromise the ability of GAP to trigger GTP hydrolysis) Catalytic cleft of GTPase activity  PI3K is another effector of RAS and RTK RAS interacts directly with the catalytic structure of PI3K  PI3K-AKT pathway Arachidonic acid Fatty acid Phosphatidylinositol 3-kinase (PI3K), a lipid kinase PIP3 recruits the serine/threonine kinase PDK-1 to the membrane. AKT, another serine/threonine kinase, is also recruited to the membrane where it is phosphorylated and activated by PDK-1. PDK1 (3-Phosphoinositide-dependent kinase 1) AKT activates downstream signaling Glucose metabolism Proliferation, Survival, anti-apoptosis Cell cycle regulation Ribosome biogenesis translation  AKT activates mTOR mTOR (Mammalian target of rapamycin) a serine/threonine kinase, a downstream target of AKT involved in promoting anabolic programs such as lipid and nucleotide synthesis.  intracellular tyrosine kinase Src Upon stimulation of EGFR by growth factor, the autophosphorylated receptor can interact with the SH2 domain of Src, disrupting its negative regulatory intramolecular conformation. Focal adhesion kinase (FAK) can also activate Src by direct binding of FAK to the SH2 domain of Src. Full tyrosine kinase activity of Src only occurs following a final p-Y416, removing obstructing activation loop from catalytic cleft.  Src signaling Activation of Src leads to disassembly of focal adhesions and thereby permits increased motility. Src also regulates cell invasion by inhibiting E-cadherin, and influences proliferation and survival. Assembly of focal adhesions facilitates cell adherence, while disassembly facilitates motility. Cell cycle regulation by integrin-mediated adhesion  The Myc oncogene c-Myc is a transcription factor that is constitutively and aberrantly expressed in over 70% of human cancers (two homologues: N-Myc, L-Myc) c-Myc is a basic-helix-loop-helix-leucine zipper transcription factor The binding of the c-Myc/Max dimer to E-box DNA can activate a large cohort o gene transcription (>1000)  Timeline of Gleevec Bcr-Abl protein functions as a constitutively activated tyrosine kinase, similar to the Abl oncoprotein of Abelson mouse leukemia virus. By 1990, Bcr-Abl cDNA was introduced into a retrovirus vector and the virus was found to induce mice leukemia resembled human CML. In early 1990, low-molecular weight antagonist of the Bcr-Abl tyrosine kinase activity was investigated. Imatinib mesylate (Gleevec, STI571) was discovered. It binds the catalytic cleft and stabilizes a catalytically inactive conformation of Bcr-Abl.  Therapeutic strategies that target protein kinases  Cetuximab (Erbitux ) and panitumumab (Vectibix ; ABX-EGF) are monoclonal antibodies that target EGFR and have been approved to treat colorectal cancers. Patients with KRAS mutations in their tumors do not respond to cetuximab (Erbitux ) and panitumumab (Vectibix ). In 2009, the US FDA updated the cetuximab (Erbitux ) label to include a recommendation on screening for the K-RAS mutation. The influence of genetic information on an individual’s response to a drug, pharmacogenomics, helps doctors to choose the best treatment for an individual  Cell cycle checkpoints Checkpoints consist of biochemical signaling pathways that induce cell cycle arrest in response to insults that threaten cell integrity and homeostasis.  Regulation of cell cycle entry (G1 checkpoint) increased expression of cyclin D increasing activity of cyclin D–CDK4/6 complexes phosphorylates and slight inhibits RB, causing moderate E2F activation The balance shifts from an inhibited E2F upregulates cyclin E expression, which binds cell cycle state to a fully active and activates CDK2. state, passing the restriction point. newly formed cyclin E–CDK2 complexes also phosphorylate RB to further activate E2F ----- a positive feedback loop The growth factors that provided the initial stimulus are now no longer required for cell cycle progression.  The G2 checkpoint The G2 checkpoint blocks entry into M phase in cells that have incurred DNA damage in previous phases or have not correctly completed S phase Upon sensing DNA damage, the cell halts the cell cycle and activates DNA repair mechanisms. DNA damage activates ATM and ATR (two upstream kinases). ATM preferentially phosphorylates and activates the kinase CHK2. ATR phosphorylates and activates CHK1 kinase CHK1 inhibits CDC25, such that it is unable to remove the inhibitory phosphate groups on CDKs that were added by the kinase WEE1. CHK2 activates p53 to induce transcription of the gene encoding p21. p21 directly binds and inhibits cyclin B–CDK1.  The mitotic (M) checkpoint During M phase, sister chromatids are separated by the mitotic spindle. The mitotic checkpoint (i.e. the spindle assembly checkpoint) is a signaling cascade that ensures correct chromosomal segregation during mitosis and the production of two genetically identical nuclei. This checkpoint monitors whether all sister chromatids have successfully attached to the mitotic spindle and whether the mitotic spindle is intact and functional.  tumor suppressor p53: Master Guardian and Executioner The TP53 gene was the first tumor suppressor gene identified. p53 is at the heart of the cell’s tumor suppressive mechanism Nickname: “guardian of the genome.” p53 pathway is altered in most human cancers.  p53 is a transcription factor The TP53 gene, located on chromosome 17p13, contains 11 exons that encode a 53-kDa phosphoprotein. The p53 protein is a transcription factor containing four distinct domains: the amino-terminal transactivation domain, the DNA-binding domain containing a zinc (Zn2+) ion a tetramerization domain a carboxy-terminal regulatory domain  p53 is a transcription factor The p53 halts cell cycle advance in response to DNA The p53 protein binds as a tetramer to a damage and attempts to aid in the repair process. p53 response element Regulation of p53 protein by MDM2 The MDM2 protein, a ubiquitin ligase, is p53 protein main regulator. MDM2 modifies the carboxy-terminal domain of p53 and thus targets it for degradation by proteosomes in the cytoplasm. Upstream: molecular pathways of p53 activation p53 becomes activated depends on the nature of the stress signal. The upstream activators of p53 utilize three main independent molecular pathways to signal cellular distress All pathways disrupt the interaction of p53 with MDM2.  DNA damage activated kinases phosphorylate and stabilize p53 ATM and ATR directly or indirectly phosphorylate amino-terminal sites of p53, and this phosphorylation interferes with binding of MDM2. p14ARF functions by sequestering MDM2 to the nucleolus of the cell Inactivation of the p16INK4A/p14ARF locus by genetic mutation or epigenetic promoter methylation has been detected in many tumors. Many cancer cells retain wild-type p53 have eliminate p53 functions by inactivating the two copies of p16INK4A/p14ARF  Li–Fraumeni syndrome Li–Fraumeni syndrome is predominantly characterized by a germline mutation of the TP53 gene and leads to predisposition to a wide range of cancers. Patients have a 25-fold increased risk of developing cancer before they are 50 years old. The young age at which individuals develop cancer and the frequent occurrence of multiple primary tumors in individuals are characteristic features of the syndrome. Haploinsufficiency of TP53 in LFS is sufficient for tumor formation. TP53 mutation in cancer does not fit Knudson’s two-hit hypothesis.  Strategies that aim to activate endogenous p53 the development of inhibitors of the p53–MDM2 interaction: inhibitors (nutlins) are best designed to mimic amino acids of p53 These results include triggering p53 activation and its biological responses in cancer cells containing wild-type p53. Idasanutlin (RG7338; Roche) is currently in Phase III clinical trials for acute myeloid leukemia. nutlins compete with p53  Apoptosis is a programmed cell death Apoptosis is characterized by cell shrinkage, chromatin condensation (pyknosis) during early stages and membrane blebbing and nuclear fragmentation (karyorrhexis) at later stages. Eventually, the cell disintegrates into apoptotic bodies which are engulfed and digested by phagocytic cells due to exposure of phosphatidylserine on the cell surface. healthy apoptotic Apoptotic bodies: the condensed hulks of cells that remain after all the destruction has Lymphocytes occurred.  Apoptosis vs. Necrosis Differences between apoptosis and necrosis Apoptosis Necrosis Cell shrinkage and membrane blebbing Cell swelling No leakage of cell contents → phagocytosis of Leakage of cell contents → apoptotic bodies causes inflammation Triggered by intrinsic Triggered by external factors factors Chromatin condensation and precise cleavage of DNA DNA degradation  Intrinsic apoptotic pathway The intrinsic, or mitochondrial, pathway is triggered by internal stress stimuli, such as DNA damage, growth factor deprivation, or endoplasmic reticulum stress. Activation of the intrinsic pathway results in mitochondrial outer membrane permeabilization (MOMP), leading to the release of several proapoptotic factors from the mitochondrial intermembrane space into the cytoplasm, including cytochrome c and Smac/DIABLO. Cyto. C then associates with apoptotic protease- activating factor (Apaf-1) and initiator caspase-9 to form the heptameric apoptosome in an ATP-dependent fashion. This results in activation of caspase-9, which subsequently cleaves caspase-3 and other effector caspases.  Under normal condition, anti-apoptotic proteins inhibit apoptosis The proapoptotic proteins Bax and Bak are required for induction of MOMP but are kept in check by binding to antiapoptotic family members such as Bcl-2 or Bcl-XL. When antiapoptotic proteins are present in sufficient concentrations, they sequester pro- apoptotic proteins and inhibit apoptosis.  Under stress condition, pro-apoptotic proteins are activated Stress signals such as DNA damage induce activation of proapoptotic BH3-only proteins such as p53-upregulated modulator of apoptosis (PUMA), Bim, or Noxa through transcriptional or post-transcriptional regulation. The BH-3 only proteins (Bid, Bim, Noxa, Bad, PUMA): 1. Binding to the pore-forming proteins directly and stimulating homo- oligomerization, promoting MOMP or 2. Binding to and inhibiting anti-apoptotic Bcl-2 family proteins.  Bcl-2 family Induction of MOMP is tightly regulated through interactions between members of the B-cell lymphoma 2 (Bcl-2) protein family, a family of over 20 proteins. Bcl-2 family proteins share homology in at lease one out of four conserved Bcl-2 homology (BH) domains and can be grouped into proapoptotic and antiapoptotic proteins.  Two groups of Caspases Two functional group of caspases: Initiator caspases: trigger the onset of apoptosis by activating the caspase cascade (caspase 2, 8, 9, 10) Executioner caspases: destroy critical components of the cells (caspase 3, 6, 7)  IAP Inhibitors of apoptosis proteins (IAPs) block caspase action in two ways: Bind to caspases directly and inhibit their proteolytic activity In certain cases, IAPs mark caspases for ubiquitination and degradation. The X chromosome-linked member XIAP is one member of the IAP family that directly binds to, and inhibits, the activity of caspase-3 and caspase-7 after they have been processed, by binding to their active site. XIAP also inhibits caspase-9 by binding to monomeric caspase-9 and locking the active site in an aberrant conformation. NF-κB, a major transcription factor in inflammation, is a potent inhibitor of apoptosis. It induces the transcription of IAPs.  The extrinsic pathway of apoptosis FASL Death signals, TNF (tumor necrosis factor) and FAS Ligand, activate their death receptors TNF receptor and FAS receptor, respectively. Fas ligand (FasL or CD95L or CD178) is a type- II transmembrane protein expressed on cytotoxic T lymphocytes and natural killer (NK) cells. Its binding with Fas receptor (FasR) induces programmed cell death in the FasR-carrying target cell. Fas ligand/receptor interactions play an important role in the regulation of the immune system and the progression of cancer. Upon ligand binding, receptors undergo a conformational change, form homotrimers, expose death domains located at the cytoplasmic tail. BID links the intrinsic and extrinsic pathways of apoptosis. Convergence of intrinsic and extrinsic apoptotic pathways 1. Bid Caspase 8 cleaves and activated Bid. Activated tBid moves from cytosolic location to mit. and initiates MOMP. 2.Cytotoxic cells dispatched by the immune system Cytotoxic cells attach to the surfaces of targeted cells, introducing granzyme B molecules into cells which cleave and activate procaspase 3 and 8.  Apoptotic drugs  Targeting TRAIL and its receptor TRAIL: TNF-related apoptosis-inducing ligand, a cytokine of the TNF superfamily TRAIL is expressed by immune cells as a homotrimeric type 2 transmembrane or soluble protein. Approximately 80% of cancer cell lines are sensitive to TRAIL ligand and can be induced to undergo apoptosis. Apoptosis induced via a death receptor is thought to be independent of p53. Many cancers with inactivated p53 mutations may still be vulnerable to such an approach. TRAIL selectively induces apoptosis of a variety of tumor cells and transformed cells, but not most normal cells. TRAIL is expressed on different cells of the immune system and plays a role in both T-cell- and natural killer cell-mediated tumor surveillance.  Apoptotic drugs  Targeting TRAIL and its receptor TRAIL-based cancer drug candidates mainly include recombinant TRAIL ligands and DR4/DR5 receptor agonists. One recombinant human TRAIL ligand, called dulanermin, is composed of the receptor binding domain of TRAIL (amino acids 114–281): promising anti-tumor activity in animal models, testing in Phase III combination trials. Using TRAIL receptor agonistic monoclonal antibodies (e.g. mapatumumab, lexatumumab) that recognize the extracellular domain of the receptor is another strategy that has been tested in clinical trials.  Drug strategies that target the BCL2 family of proteins first approved direct apoptosis-targeting cancer drug Therapeutic agents are shown in red. Both venetoclax and SAHA (Vorinostat) are approved.  Drug strategies that target IAPs and indirectly activate caspases  Indirect method to activate caspases: Targeting IAPs SMAC mimetics are synthetic mimics of the endogenous inhibitor SMAC/DIABLO and similarly bind to the Baculovirus IAP repeat (BIR) domains of IAPs that inhibit caspases. One BIR domain of XIAP directly blocks the active site of caspase-3 and caspase-7, while another distinct BIR domain inhibits caspase-9.  Drug strategies that target IAPs and indirectly activate caspases  Indirect method to activate caspases: Targeting IAPs The small-molecule inhibitor AT-406 binds to the XIAP BIR domain known to block the active site of caspase-3 and caspase-7, the downstream caspases. Birinapant (TL32711) is a bivalent SMAC mimetic that targets two BIR domains and is being tested in Phase I and II clinical trials for ovarian cancer. IAP antisense oligonucleotides, such as GEM640 (AEG35156), have been designed to reduce XIAP mRNA and protein but have had mixed success thus far in different cancers (acute myeloid leukemia versus pancreatic cancer). Practice questions 1. In chromothripsis, what is the likely cause of the sudden genomic rearrangement? a) Exposure to mutagenic chemicals b) Errors during DNA replication c) Viral infections d) Simultaneous breakage of multiple chromosomes 2. What is kataegis? a) A type of chromosomal rearrangement b) A localized hypermutation phenomenon c) Spontaneous breakage of chromosomes d) Inversion of DNA strands 3. Nucleotide excision repair (NER) is a mechanism specifically designed to repair: a) Single-strand breaks in DNA b) Mismatches in DNA sequences c) Thymine dimers and other bulky lesions d) DNA replication errors 4. Which of the following is a characteristic feature of glycosylase enzymes? a) They add nucleotides to the DNA strand b) They break phosphodiester bonds in the DNA backbone c) They hydrolyze DNA crosslinks d) They flip out damaged bases for excision. 5. Alkylating agents, such as cyclophosphamide, work by: a) Inhibiting topoisomerase enzymes b) Cross-linking DNA strands c) Interfering with microtubule dynamics d) Blocking cell cycle progression 6. The Rb protein becomes inactive when phosphorylated by: a) cyclin A-CDK2 b) cyclin D-CDK4/6 c) cyclin E-CDK2 d) cyclin B-CDK1 7. The primary mechanism of action of nutlins in cancer involves: a) activation of cyclins b) inhibition of p53 c) stabilization and activation of p53 d) promotion of MDM2-mediated degradation Short answer question: PARP inhibitors are used to treat BRCA1/BRCA2 mutated cancers. Please provide an explanation on the underlying mechanism (10 points).