Lecture 1 (slide 1-75)

genes that regulate cell division

proto oncogene

mutated or over-expressed proto oncogenes

oncogenes

Chromosomal rearrangements

Mutations

epigenetic activation

Drie grote mechanismen van oncogene activation

Whole chromosome / segmental gain

Constitutive overexpression due to amplification

fusion/hybrid genes due to chromosomal rearrangement

constitutive overexpression due to promotor/enhancer hijacking

4 belangrijkste subtypes chromosomal rearrangements

activating mutations

promotor / enhancer mutations

mutations inhibiting degradation

oncogenic splice variants

belangrijkste mutaties die kunnen leiden tot oncogene activation

Numerical and structural rearrangements

twee types chromosomal aberrations

genomic instability

Voor welke cancer hallmark zullen Numerical and structural rearrangements zorgen?

B-cell lymphoma

Typische kanker waarbij whole chromosome gains gebeuren

c-MYC on chromosome 8

belangrijk oncogene met zijn chromosoom bij b-cell lymphoma

q24 of chromosome 8 (long arm, segment 24)

locatie c-MYC

Segmental: only a segment of the chromosome is gained or lost

wat maakt segmental chromosome gains anders dan whole chromosome gains?

• pediatrische tumor van het sympathisch zenuwstelsel

• ontstaat vaak in of rond de bijnier

• geassocieerd met recurrente segmentale chromosomale afwijkingen

• belangrijk voorbeeld: MYCN-amplificatie

Principe:
Neuroblastoma is een pediatrische tumor waarbij chromosomale gains/losses en vooral MYCN-amplificatie belangrijk zijn voor tumorbiologie en prognose.

neuroblastoma

MYCN: oncogene (N = neuronal), can be gained or amplified (on chromosome 2)

belangrijk oncogene bij neuroblastoma

1p-del

MNA

11q-del

17q-gain

typische chromosomal aberrations bij neuroblastome

small circular DNA fragments, present in the nucleus but not part of chromosomes, often highly abundant in cancer cells (can carry amplified oncogenes and contribute to high expression)

extrachromosomal DNA en kanker

chromosomal DNA

chrDNA

extrachromosomal DNA

ecDNA

chrDNA: higher order compaction

ecDNA: lower order compaction (strong expression)

chrDNA vs ecDNA op basis van compactheid en expressie

ecDNA lacks a centromere → no proper segregation during cell division → random distribution over daughter cells → can be lost

is ecDNA stabiel?

To stabilize amplification, ecDNA can re-integrate into chromosomes → forming HSR (homogeneously staining regions)

methode ecDNA om stabieler te zijn

EGFR (lung cancer)

ERBB2/HER2 (breast cancer)

MYCN (neuroblastoma)

DHFR (leukemia)

belangrijke oncogenen die vaak gelinkt zijn met ecDNA of HSR

• Oncogene amplification

  • ecDNA draagt extra kopieën van oncogenen

• Chromatin decompaction

  • ecDNA is vaak opener/losser verpakt → hogere expressie

• Enhancer hijacking

  • ecDNA kan oncogenen samenbrengen met sterke enhancers

• Chromosomal reintegration

  • ecDNA kan opnieuw integreren in chromosomen → HSR

• Intra-tumor heterogeneity

  • ecDNA wordt ongelijk verdeeld bij celdeling → verschillen tussen tumorcellen

• Drug resistance

  • ecDNA-copy number kan snel veranderen onder therapiedruk

Principe:
ecDNA versterkt oncogene expressie en maakt tumorcellen genetisch flexibeler, wat heterogeniteit en therapieresistentie bevordert.

6 modes of action van ecDNA

relatively simple rearrangement → duplication of a DNA segment, resulting in multiple adjacent copies of the same region (can include oncogenes)

tandem duplication

Tandem duplicator phenotype

TDP

ovarian cancer (OV)

triple-negative breast cancer (TNBC)

waar vind je de hoogste TDP rates?

reciprocal translocation

not loss or gain, but exchange of DNA between chromosomes

(no net gain or loss of genetic material)

fusion / hybrid genes

mogelijk gevaarlijk gevolg van reciprocal translocation

t(9;22)(q34;q11) translocation and the BCR/ABL1 fusion oncogene in CML (chronic myeloid leukemia)

Philadelphia chromosome

BCR and ABL close to each other, leading to loss of regulation of kinase activity, leading to constant phosphorylation. → increased proliferation and survival of leukemic cells

effect philadelphia chromosome

acute lymphoblastic leukemia

ALL

breakpoint cluster region

BCR

Abelson Murine Leukemia Viral Oncogene Homolog 1

ABL1

Chronic myeloid leukemia

(clonal hematopoietic stem cell disease )

CML

• één hematopoëtische stamcel krijgt een driver-mutatie

• deze kloon breidt uit en maakt te veel myeloïde cellen

• in chronische fase blijven die cellen vaak nog deels functioneel

Principe:
CML start als clonale expansie van myeloïde cellen, maar kan later evolueren naar blast crisis met meer immature, minder functionele cellen

gevolg clonal hematopoietic stem cell disease

Evolvement to fatal acute leukemia through a so-called acceleration phase

additional karyotypic abnormalities (+8, i(17q), +Ph, +19) and transcriptome changes

= BLAST CRISIS

hoe van clonal hematopoetic stem cell disease naar fatal acute leukemia?

BCR-ABL1 on its own is sufficient to maintain the leukemia phenotype of CML

oncogene addiction in CML

mitogen-activated protein kinase

MAPK

Normally ABL1 shuttles between nucleus and cytoplasm, but now it stays in the cytoplasm

impaired shuttling bij BCR-ABL1

MAPK: increased proliferation

JAK-STAT pathway: impaired transcriptional activity

PI3K / AKT pathway: increased survival, proliferation

activation of c-MYC

gevolg van impaired shuttling bij BCR-ABL1 op pathways

Mitogen-activated protein kinase

MAPK

phosphoinositide 3-kinase

PI3K

gleevec

drugs dat werkt tegen BCR-ABL

goes into competition with ATP, which can’t attach to the kinase

werking Gleevec

Imatinib

Gleevec is brand name of …

40 different mutations in amino acids in the tyrosine kinase domain leading to resistance

resistentie tegen imatinib

T315I

typische puntmutatie dat zorgt voor imatinib resistance

Imatinib

dasatinib

nilotinib

ponatinib

(=> ImDaNiPo)

second generation inhibitors van BCR/ABL1 oncoprotein

can also bind to ATP-pockets of tyrosine kinase receptors with similar structural features as ABL1

extra toepassing Gleevec

germline mutations in c-KIT en PDGFRA in vormen van GIST

waar kan gleevec nog werken?

KIT Proto-Oncogene, Receptor Tyrosine Kinase

c-KIT

gastro-intestinal stromal tumors

GIST

t(15;17)(q22;q22) translocation PML-RARA oncogene in PML

fusion protein met PML

promyelocytic leukemia protein: organiser of nuclear subdomains (“PML bodies”)

PML

PML-RARA blocks differentiation of bloodcells, and lead to accumulation of immature cells

gevolg PML-RARA fusion protein

ATRA (all trans retinoic acid)

therapie tegen PML-RARA

ATRA restores differentiation by releasing transcriptional repression

ATO promotes degradation of the PML-RARA fusion protein

combined ATRA-ATO therapy

Arsenic trioxide

ATO

Arsenic trioxide induces sumoylation and ubiquitination → degradation of PML-RARA

hoe zorgt ATO voor degradatie van PML-RARA?

targeted protein degradation

van wat soort behandeling is ATRA-ATO het eerste voorbeeld?

PROTAC

latere drugs voor targeted protein degradation

Right: ligand recruiting E3 ligase

Middle: linker

Left: ligand binding the target protein (e.g. androgen receptor) → induces degradation of the target protein

onderdelen PROTAC

t(11;22) to form EWS- FLI1

fusion genes bij Ewing sarcoma

solid tumour, sarcoma in bone or soft tissue; this example in bone

Wat voor tumor is Ewing sarcoma?

EWS provides a transactivation domain

FLI1 provides a DNA-binding domain (ETS transcription factor)

Fusion → aberrant transcription factor with constitutive activity

gevolg EWS-FLI1 fusion

You’re not born with the fusion product. There is a certain window when the fusion product leads to transformation. When made too early or too late, it won’t happen

Developmental window for fusion genes driven transformation

In the experiment, in the different stages they use different promotors. They choose a promotor that is active in a specific stage, link it to the fusion gene, and check if it is transformative

Lethal: it is too early

experiment om te testen op developmental window

TMPRSS2-ERG

belangrijke fusie bij prostate cancer

androgen responsive transmembrane serine protease

TMPRSS2

kinases or transcription factors

belangrijke zaken die vaak in fusion genes zitten

many translocations forming fusion genes or mediating promotor/enhancer hijacking are pathognomonic for specific cancer (sub) entities

RNA-sequencing can detect reads spanning the fusion junction, allowing identification of fusion genes and patient classification

klinische relevantie fusion genes

finding that is highly specific for a certain cancer entity; if that fusion product is present, it strongly points to that specific diagnosis

pathognomonic

chromothripsis

a catastrophic one-time chromosomal event in which a chromosome (or part of it) breaks into many fragments and is incorrectly reassembled.

complex chained rearrangements involving multiple chromosomes

chromoplexy

circos plot
Method to comprehensively show genomic alterations in a tumor cell

wat is dit?

a strong enhancer is brought near another gene promoter, activating that gene

enhancer hijacking

TCRα/δ and TCRβ loci contain strong enhancers

Translocation brings the TLX gene close to a TCR enhancer (not a fusion protein, but enhancer hijacking)

TLX is normally not expressed in these thymic cells, but becomes highly transcribed after the translocation

gevaar bij T cell receptor (TCR) encoding loci in context van ALL

High TLX expression causes maturation arrest and drives TLX-addicted leukemia

gevolg hoge TLX expressie

IGH/BCL2 rearrangement

BCL2 stops apoptosis

belangrijke translocation bij follicular lymphoma

Intrinsic / mitochondrial pathway releases cytochrome c, forming apoptosome. This release is blocked by BCL2

hoe is BCL2 anti-apoptotic?

Tyrosine kinases (transmembrane receptors) that display kinase activity

Dimerize after ligand binding, bringing the tails close together, leading to phosphorylation of each other

The phosphorylated residues are docking sites for adaptor proteins, which activate downstream signaling

werking principe growth factor receptor signaling

PDGRF:

belangrijk voorbeeld growth factor receptor

platelet derived growth factor receptor

PDGRF

>25 yrs ago discovered as oncogenes, more recently as targets for therapy

belang growth receptors in context van kanker

strictly regulated: low conc. phosph tyrosine proteins in non-dividing cells

hoe sterk zijn growth receptors gereguleerd?

src

PI3K

belangrijke adaptor proteins bij de PDGFR

Proto-Oncogene, Non-Receptor Tyrosine Kinase

wat voor iets is SRC?

each level of regulation can be deregulated: ligand, receptor (eg more receptors), downstream signaling (also by point mutations), transcription factors, target genes

wat kan gedereguleerd worden bij de growth receptors?

Lung cancer

waar is de receptor tyrosine kinase: EGFR belangrijk?

breast cancer

waar is re receptor tyrosine kinase, ERBB2 belangrijk?

amplification of ligand, receptor or point mutations for constant activation (ligand-independent firing)

Point mutations can make the receptors dimerize / activate without ligand

Loss of the extracellular part can also cause constitutive activation

methoden van overexpressie bij growth receptors

• Regulatory type mutations

  • mutaties in regulerende domeinen

  • halen de “rem” van de receptor weg

  • → ligand-onafhankelijke/constitutieve activatie

• Enzymatic pocket mutations

  • mutaties in het kinase/ATP-bindend domein

  • veranderen kinaseactiviteit of drug binding

  • → belangrijk voor gevoeligheid/resistentie tegen TKIs

Principe:
c-KIT kan oncogeen worden door verlies van regulatie of door veranderingen in de enzymatische kinase-pocket

belangrijke klasses van (c-KIT) mutaties

EGF → EGFR → GRB2 / SOS → RAS (KRAS) → RAF → MEK → ERK → nucleus → proliferatie

belangrijke cascade dat volgt op binding van EGF op EGFR receptor (bij lung cancer belangrijk)

Signal enters the nucleus through ERK

belang ERK

Smokers: more KRAS

Non-smokers: more EGFR

=> KRAS and EGFR mutations are mutually exclusive, (so or one or the other)

resp in smokers and non-smokers

NF1 is a tumor suppressor (loss-of-function), while the others are mainly activating alterations

belangrijke drivers die gemuteerd worden bij lung cancer

maken de receptor constitutief actief, waardoor proliferatie- en overlevingssignalen blijven doorgaan.

gevolg punt mutaties / deleties in EGFR

Erlotinib

Gefitinib

Voorbeelden als therapieën bij long kanker, specifiek tegen EGFR

Range where tumor cells are inhibited while WT cells are relatively spared.

therapeutic window

T790M mutation increases ATP affinity and requires higher inhibitor concentration than WT EGFR, causing loss of the therapeutic window and toxicity

resistentie tegen first generations EGFR TKI

Gefitinib

voorbeeld first generation EGFR TKI

stronger inhibition of mutants (including T790M), but WT EGFR is still inhibited too much, so limited therapeutic window

probleem second generation EGFR TKIs