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Exam 1 Details

• Exam date is 2/17/25 (Monday)
• Exam takes place from 10:00 am - 11:15 am
• The exam will be in S 105 (in-person in-class in Canvas)
• Lecture material covers lectures 1-8
• Update the lockdown browser before the exam
• Check out after submitting the exam before leaving the classroom
• The exam format includes 45 multiple choice questions, each worth 2 pts
• There will be 1 short answered question, worth 10 pts

Cancer Terms

• Carcinomas are malignant tumors of the epithelium
• Sarcomas are malignant tumors of the mesenchyme, such as bone or muscle
• Adenocarcinomas are malignant tumors of a gland, such as breast cancer
• Angiogenesis is the formation of new blood vessels
• Metastasis is the process of cancer cells spreading from a primary site to secondary sites.

Types of DNA mutations

• Transition is the substitution of one purine for another purine
• Transversion is the substitution of a purine for a pyrimidine or vice versa
• Insertion involves adding a base
• Deletion involves removing a base
• Chromosomal translocation involves swapping genetic material between chromosomes

DNA Mutations

• Additional mutations have been identified from recent advances in sequencing technology and imaging
• Kataegis, Greek for "thunderstorm," describes small localized areas of hypermutation
• Chromothripsis, from "chromo" meaning chromosome and "thripsis" meaning breaking into small pieces, is a one-off cell crisis resulting in tens to hundreds of genomic rearrangements

Chromosomal and circular extrachromosomal DNA

• Tumor cells contain large circular ecDNAs, ranging from 100 Kb to 1.3 Mb, found in one in four solid tumors but rarely in normal cells
• ecDNAs can replicate without centromeres resulting in unequal segregation to daughter cells during cell division Leanding 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 does not reside on a chromosome, is commonly observed in human cancer cells, and carries oncogenes

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
• This is minimized by exonuclease proofreading activity of replicative DNA polymerases and post-replication surveillance by the mismatch repair (MMR) system
• Failure of repair mechanisms in tumors is associated with a very high mutation burden described as "hypermutation" or "ultramutation"

Mechanisms maintaining genomic stability

• The fidelity of DNA duplication is further increased by the mismatch repair (MMR) system
• MMR corrects replication errors that have escaped editing by polymerases
• MMR consists of base-base mismatches, as well as insertions, and deletions produced as a result of slippage during the replication of repetitive sequences described as microsatellites
• Highly repeated sequences in the genome, carrying 100 or more nucleotides per repeat unit, “satellite” sequences
• Far shorter repetitive sequences found in many places in the genome have been named "microsatellite"

Mismatch repair deficiency (Lynch syndrome)

• Lynch syndrome, also known as hereditary non-polyposis colon cancer or HNPCC
• HNPCC is an autosomal dominant condition predisposes sufferers to various malignancies including colorectal, ovarian, endometrial, stomach, and prostate cancer
• HNPCC responsible for 2 to 3% of all colon cancer cases
• The majority (85-90%) of HNPCC cases result form 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%)

Mismatch repair mechanism-lecture 2

• 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 with microsatellite repeats leading to microsatellite instability and a very high mutation rate overall
• Most Lynch syndrome-associated tumors display microsatellite instability, particularly in cases with MSH6 variants

Polymerase Proofreading Domain mutations

• 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 (exonuclease) domains of PolD1 and PolE genes have been observed in endometrial cancer, colon cancer and other tumor types
• Tumors with these mutations lead to ultra-mutated phenotypes with as many as one million base substitutions in individual tumors
• In contrast to MMR-deficient cancers, 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 leads to dysregulation of mutations
• APOBEC is a cytidine deaminase family that plays important roles in pathogen defence by inducing DNA damage of infecting RNA and DNA viruses, and in innate immunity
• It aids antibody diversification by causing somatic hypermutation (C to T mutations) at the antigen-binding sites, but can also lead to C>T and C>G mutations from DNA repair intermediates causing genetic instability in human cancers
• APOBEC overexpression leads to kataegis and cancer and drives cancer drug resistance, accounting for the majority of mutations in 10% ER+ breast cancers

Summary of DNA repair pathways

• DNA damaging agents like ionizing radiation, X-rays, and anti-tumor drugs create double strand breaks, single strand breaks, intrastrand and interstrand crosslinks
• These are repaired by Non Homologous End Joining or Homologous Recombination, with key mutated proteins BRCA1/BRCA2
• UV light chemicals produce bulky adducts and pyrimidine dimers, which are repaired by Nucleotide Excision Repair, and key mutated proteins include XP and ERCC
• Oxygen radicals and alkylating agents can lead to abasic sites and 8-oxoguanine lesions, which are repaired by Base Excision Repair, with key mutated proteins MUTYH and PARP
• Replication errors can lead to bases mismatch, insertions, and deletions, which are Repaired by Mismatch Repair and key mutated proteins include MSH2, MLH1, MSH6, and PMS2

Comparison of three repair mechanisms

• Base excision repair removes oxidation, deamination, and alkylation and is involved in the removal of spontaneous damage by glycosylase
• Key mutated proteins are OGG1 MUTYH PARP
• Mismatch repair corrects DNA copy errors
• Key mutated proteins are MSH2, MLH1, MSH6, PMS2 _ Nucleotide excision repair removes chemicals and thymine dimers from radiation
• Key mutated proteins are XP ERR

Targeting PARP in BRCA mutant cancer patients

• PARP is targetted in BRCA mutant patients, using synthetic lethal interactions In the face of DNA damage normal cell SSB's are repaired, in cells with double break strands BRCA is activates to repair (HR-proficient) to maintain cell survival, in some cases a Cell survival is needed
• Cancer cells, that contains BRCA as damaged cells cannot carry out BRCA mutations, cells undergo a synthetic lethality

Nucleotide excision repair defects (XP)

• Xeroderma pigmentosum (XP) syndrome
• • In 1874, 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 with minimal sun exposure
• XP individuals have 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

Base Excision Repair Defects

• A family of DNA damage-specific glycosylases (such as OGG1 and MUTYH) initiate BER
• Inherited mutations in MUTYH cause multiple colorectal adenoma syndrome also is known MUTYH-associated polyposis (MAP)
• MAP patients have increased risk colorectal cancers
• Patients with MAP mutations have a lifetime risk of colorectal cancers
• Indivuals with MAP may develop upper gastrointestinal tract and cutaneous tunnels

Chemotherapy

• The three main types of chemotherapy are:
• Alkylating agents and platinum-based drugs
• Antimetabolites
• Organic drug

Chemotherapy: Alkylating agents and platinum-based drugs

• Alkylating agents includes:
• Nitrogen mustards including cyclophosphamide, ifosfamide, melphalan, chlorambucil
• Nitrosoureas carmustine, loumustine, streptozotocin
• Alkyl suphonate busulfan
• The mechanism of action is to alkylate the N7 residue of the guanine by crosslinking nucleobases in DNA double-helix strands
• Platinum-based agents, including cisplatin, carboplatin, oxaliplatin
• Form covalent bonds via the platinum atom and bind N7 guanine, leading to intra- and interstrand DNA crosslinks that inhibits DNA synthesis and inducing programmed cell death

Chemotherapy: Antimetabolites

• Agents resemble an endogenous metabolite that blocks 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 synthes
• Antifolate analogues (methotrexate, pemetrexed) impair the function of folic acids
• Methotrexate is a folic acid analogue, that binds and inhibits dihydrofolate reductase (DHFR)and prevents tetrahydrofolate formation essential for purine and pyrimidine synthesis and protein production (Ser and Met synthesis)

Chemotherapy: Organic drugs

• Anthracyclines (doxorubicin, daunomycin, epirubicin, daunorubicin) are antitumor antibiotics derived from the Striptomyces bacteria Three mechanism of actions:
• Inhibit DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand, preventing the replication of rapidly-growing cells
• Topoisomerase II enzyme is inhibited, preventing the relaxing of super-coiled DNA and blocking DNA transcription and replication
• Iron-mediated free oxygen radicals that damage the DNA and lipid domain of cell membranes are created Cardiac damage is its most severe side effect, treat solid tumors (e.g. of the breast or lung)

Chemotherapy Drugs: Microtubule inhibitors

• Vinca alkaloids (vincristine, vinorelbine, vinblastine, vindesine) is an antimitotic and antimicrotubule agents
• Originally derived from the Periwinkle plant Catharanthus roseus
• Leads to principle mechanisms of cytotoxicity:
• Microtubule function is disrupted, particularly the microtubules comprising the spindle apparatus leading to metaphase arrest.
• Taxanes (docetaxel, paclitaxel, nab-paclitaxel) are diterpenes produced by the plants of the genus txus (yews) Principle mechanism of action:
• Taxnes Disrupt microtubule funtion, disrupt of cell division, stabalize GDP by tubin resulting in cell death Taxanes are mitotic and destroy spindles during cell divison They are called spindle poisionies

Mechanisms of drug resistance

• Cancerous cells acquire resistance to Anticancer drugs, = Drug
• Modify drug metabolism to resist anticancer drugs Increasing a eflux and decrasing the intake of drug Increasing the number of target molecules and alltering metabolism Altering DNA repair mechanisms to resist drug intake

The efflux

• Multi-drug resistance expression of ATP-dependant efflux drives resistance, as well as P-glycoprotien P-(P-gp, ABCB1)
• P-(p multi-drug resistance gene drives expression of ATB-binding factors like Alaloids or DNA stabalizing

Super-enhancer

• Super-enhancer: Large clusters of multiple distinct TFs (each bind to their own enchancers) of nearby non-housekeeping genes
• It enables gene transition Tumorgenesis drives tumour cells to aquire ongcogenes and disregulate cell pathways

Acetutation

• Histones nuetralize lysien residues and chromatain folding
• Acetylatiol are enhance transcriptional elongation
• Lysiens are at all four core domains
• Transcription factors such as e2f P53 enhance lysien
• Kat enhances acetylate

-Ac restores the charge for Lysine and packing for high level transcription

DNA methylation

• Cytosine residues are addded to DNA methylation at 5' position with an addition of a methyl group to position 5 Methylation occurs at 3-4% of cell cytosines methylation requires cytosines at 5'-guanine (CpGs and DNA) methylation.

DNA AND CpG islands

• CpG islands are for clustered regions of the promoter Methylatio may occur Methylates cytosines are found repeatitive sequence Methylation leads to cancer and epigenitics

methyltransferase

• DNMTs: used in the conversion hemi-methylated DN to fulll methylated DN and allow mehtlaion patterns to signal to to daughter cells

Intratumor epigenetic alterations

• Aberrant dna methylation via tumours (hypermethylation- cancer, and LY64K glioblastoma and SL34A2 is papiary)
• Additional genes (functions via PTEN) are hypermethlatef

BETI

• protein inhibitors of DNA binding

Histone lysine methylation and KMT's

Histone Methyltransferase Inhibitors

• Small molecule EZH2 inhibitors, such as EPZ-6438 (tazemetostat), GSK2816126, and CPI-1205, approved
• Can cause Follicular lymphomas due to to gene repression if IRF4 IS NOT ACTIVE, if active cell will arrest

Non-coding RNA's

• Regulatory via linear and circular RNA
• Regulatory via LncRNAs, snoRnas and siRNA

Mirco RNA

• small, protein-coding RNA (18-25)
• The primary transcripts are processed to ribonucleates including the Dicr8 proteins

CIRCULA RNA

• (5' RNA)
• half lies from lack of free 3, or 5' ends
• can from continueous loops

LncRnA functions

Guide

Scaffold

Decoy

MiRrna

LncRNA exampleS

• HOTAIR (HOX anstisense intergenic RNA)

telomore

Telomeres repeats in the seqence strand

TELAMORASE

• riboprotei containing humans
• Maintains telemore length

CELL CYCLE

• The primary mechanism for Uni directional is for an organised cell cycle

Mechanisms of CDK regulation

• CDK needs activation by CAK by associating in the CDK activity
• CDK neads inhibition is to be negatively regulated by CKIs by inhibit Cdk ACTIVITY

CKIs

• Family (p) and specific for CK4
• Proteins are traned and bind CDk4 and CDDk6
• CD-cyclin and A ande/b complexes

p5K ACTIVIATION

• CdK requries phosphorylation

REPECTOR TYROSINE KINESS

EGFR ERB Reurolin, EGFR HER Her 3 weak Oncogene halagone

Grb2

• Src homology for Grb2 links
• 2SOS and guanine for nuclear
• -Intermediatate in cell switch

Ras

Catalyz the releasing

Mdm2 for p53 in cancer treatment (protein)

• A,a transcription

• Apoptosis during cancer

Practice Questions and Answers

• In chromothripsis, what is the likely cause of the sudden genomic rearrangement? Simultaneous breakage of multiple chromosomes
• What is kataegis? localized hypermutation phenomenon
• Nucleotide excision repair (NER) is a mechanism specifically designed to repair: Thymine dimers and other bulky lesions
• Which of the following is a characteristic feature of glycosylase enzymes? They flip out damaged bases for excision.
• Alkylating agents, such as cyclophosphamide, work by: Cross-linking DNA strands
• The Rb protein becomes inactive when phosphorylated by: cyclin E-CDK2
• The primary mechanism of action of nutlins in cancer involves: stabilization and activation of p53.