Neuroblastoma. 72 good questions, nicely answered!

Q: What is the SRC (Sarc) oncogene?

-SRC is one of the 4 genes encoded by the Rous Sarcoma Virus (RSV). When it is mutated in prevents sarcoma induction in chicken. - The viral SRC gene is the mutated copy of the chicken SRC gene , the virus 'high-jacked' the chicken SRC. - All vertebrates including man encode a SRC gene - WT human and mouse SRC does not induce cancer - Mutation in viral SRC are respondible for oncogenesis

Q: What are the results of the cancer genome/exome sequencing?

- Confirmation known genes & their mutation frequencies : Ras family genes, GF receptors, GFs, signal transduction genes (MAPK pathway, PI3K pathway), cell cycle genes, apoptosis genes. - Entire new group genes: Epigenetic chromatin modifiers - SWI/SNF (BAF) complex members, mutated in 20% of all tumors

What are the 2 main theories about the cause of cancer?

1. Viruses cause cancer
2. Chromosomal defects/endogenous genes cause cancer

What is the SRC (Sarc) oncogene?

-SRC is one of the 4 genes encoded by the Rous Sarcoma Virus (RSV). When it is mutated in prevents sarcoma induction in chicken.
- The viral SRC gene is the mutated copy of the chicken SRC gene, the virus 'high-jacked' the chicken SRC.
- All vertebrates including man encode a SRC gene
- WT human and mouse SRC does not induce cancer
- Mutation in viral SRC are respondible for oncogenesis

How were oncogenes identified?

1. Via Retroviruses
2. Via chromosomal translocations in tumors
3. Via cellular transformation essays

How were oncogenes identified via retroviruses?

- All cancer-inducing animal retroviruses were analysed. Many of them harbored vertebrate cDNA sequences that caused cancer --> the cellular homologues of those were identified as oncogenes.

Ki-Ras oncogene:- Transformed gene in Kirsten Rat Sarcoma 2 Virus
- Cellular homologue Ki-ras not oncogenic
- Viral Ki-ras carries oncogenic mutation

How were oncogenes identified via chromosomal translocations in tumors?

- There were recurred specific breaking points in chromosomes in tumors.
- A new gene is formed with oncogenic properties when chromosome translocation occurs.

Philadelphia chromosome:
chr.9 with chr. 22 (t(9:22)). The translocation activates the Abelson oncogene which causes chronic myonic leukemia.

How were oncogenes identified via cellular transformation essays?

Oncogenes:
- mutated oncogenes can activate cells in vitro
- oncogenes in transgenic mouse strains induce cancer
- oncogenes (in)directly regulate cell division
--> mutated oncogenes cause cancer

- Mutated oncogenes cause cancer --> cancer is caused by activated oncogenes.
- Many oncogenes have a 'logical function'

What are the options for sequencing cancer genomes?

- exons only (genes only): 1% of genome
- whole genome sequencing (WGS)

What is the 'genetic landscape/frequent mutations) of colon cancer?

- Frequently mutated: APC, K-RAS, P53
-Many genes with recurrent mutations but at low frequency

What is the TCGA Consortium: The Cancer Genome Atlas?

- Big research on the cancer genomes, a NIH initiated project
- 33 types of tumors sequences
- Tumors & normal tissue sequenced of > 11.000 patients

What are the results of the cancer genome/exome sequencing?

- Confirmation known genes & their mutation frequencies:
Ras family genes, GF receptors, GFs, signal transduction genes (MAPK pathway, PI3K pathway), cell cycle genes, apoptosis genes.

- Entire new group genes:
Epigenetic chromatin modifiers - SWI/SNF (BAF) complex members, mutated in 20% of all tumors

What is the SWI/SNF (BAF) complex?

The BAF complex is an epigenetic regulator of gene expression.

- 15 subunits
- some subunits vary (BAF 200/250A/250B)
- specific subunits mutated in specific tumors

What are the 3 generations of sequencing technology?

- 1st generation (conventional): Sanger
- 2nd generation (Massively parallel): Illumina
- 3rd generation (Single molecule)

How does bioinformatics data processing go?

1. Genome alignment & reconstruction
Mapping to reference genome (single-end, paired-end)

2. Somatic variation identificationDifference with reference genome. Always sequence both the tumor and normal DNA of patient! There are already over 3 million differences in BPs (SNPs) between people. A difference is a SNP (single nucleotide Polymorphism).
Somatic mutation = change of base with respect to normal DNA

3. Copy number identificationAmount a piece of DNA is sequenced. Log(Tumor/Lymphocytes). Identify it there is a piece extra or gone.

4. Identification structural variantsInsertions, deletions, translocations, Inversion (Paired end sequencing)

5. Condensing information for interpretation
Circus plots

What is the difference between driver and passenger mutations? (somatic mutations)

Driver mutations: confer growth advantage on cancer cell.
- functionally critical
- positively selected during evolution of the cancer
- reside in oncogenes/tumor suppressor genes

Passenger mutations: do not confer a growth advantage.
- neutral effect on cancer cell
- passenger mutations induced by e.g. Carcinogens, sunlight
- many passenger mutations in melanoma, lung cancer
- passenger mutations that cause amino acid changes can be immunogenic!

Differences: frequency (recurrence), damaging or not

What is the difference between activating mutations (oncogenes) and inactivating mutations (tumor suppressor genes)?

Both copies mutation --> tumor suppressor
One copy mutation --> oncogene

Oncogene: a specific spot for mutation
Tumor suppressor: mutation can be everywhere, while the 'activating' (oncogene) can only happen in a few specific ways. 'Breaking' (inactivating suppressor genes) can happen in many ways.

What is neuroblastoma? Where is it located?

- Neuroblastoma: childhood tumor of the peripheral sympathetic nervous system.
- Most neuroblastomas are located in the abdomen. Associate with adrenal gland/sympathetic ganglia.
- Primary tumor: Adrenal glands, peripheral nerves
Metastasis to: bone, bone marrow, liver

* High stage neuroblastoma are already metastasized at diagnosis

What is the difference between low stage and high stage neuroblastoma tumors?

- Low stage tumors: localized within adrenal glands (sometimes spinal nerve root).
- High stage tumors: metastasized disease. Primary tumor + metastatic lesion. (lymph nodes, skin, liver, bones, bone marrow)

What is the staging system of neuroblastoma?

I: no dissemination
II: loco-regional metastases
III: loco-regional metastases crossing midline
IV: distant metastases (bone, bone marrow, skull, liver)
IVS: distant metastases limited to skin & liver (spontaneous regression!)

Stages do not represent progression states! Stage III & IV diagnosed as such!

What is stage 4S neuroblastoma?

- Special stage of neuroblastoma which enters spontaneous regression
- Metastasis to liver (& skin)
- Spontaneous regression
- Wait-and-see protocol in clinic
- Good prognosis

What is the current treatment for neuroblastoma?

Diagnosis --> Chemo/Chemo/Chemo --> Surgery --> Stem cell transplantation --> Consolidation therapy

- Chemo is a cocktail of 2 different drugs
- Chemo course depends on condition patient
- Children can handle much higher chemo doses
- After surgery also a very high dose chemo (after stem cell 'harvest')

Aim:
1. Shrink tumor mass/volume (chemo)
2. Removal tumor (surgery)
3. Kill all remaining cells (consolidation therapy)

What is the consolidation therapy for neuroblastoma?

1. Differentiation therapy: Retinoic acid
2. Immune therapy: Anti-GD2 antibody

* The GD2 is also present on nerves, this therapy causes severe pain

What is clinical remission?

- No detectable cancer cells

* However, there is a detection limit --> cancer relapse

Where do nerves originate from? Describe pathway.

- the sympatho-adrenal lineage develops from the neural crest

- Cells at the top of neural crest undergo EMT and migrate downstream. There they become lineage-commited precursors (sympathoadrenal precursors) with the help of 'master genes'. After this the precursors can differentiate into adrenal chromaffin cells or sympathetic ganglia. For the differentiate into the sympathetic ganglia they need DBH and TH.
- Sympathoadrenal precursors are also adrenalin secretion cells
- Master genes = determine differentiation
- Genes sympathoadrenal precursors = PHOX2A, PHOX2B, GATA2, GATA3, SOX4, SOX11, INSMI, HALID2
- Sympathetic ganglia = DBH & TH

What are the indications that neuroblastomas are derived from sympathoadrenal precursors?

- The expression of PHOX2A and PHOX2B (master upstream regulators) and DBH (differentiation into sympathetic ganglia).
- The PHOX2A/B - DBH cascade is preserved in tumors
- Neuroblasts are precursors of adrenalin-producing cells
- Adrenalin synthesis route is preserved in tumors
- Neuroblastoma produces catecholamines

So the neuroblastomas are from the adrenergic lineage (also express the metabolites).

How is neuroblastoma diagnosed?

With MIBG scintigraphy:- differentiated neuroblasts produce adrenalin; they express adrenalin re-uptake receptors.

MIBG = meta-iodebenzylguanidine, an adrenalin-analogue
The MIBG is labeld with 123I or 131I
--> 90% of neuroblastoma is MIBG+

What are the characteristic mutations of neuroblastoma?

- Amplification MYCN oncogene:
targeted MYCN expression induces neuroblastoma.
- Mutation PHOX2B:
mutation acts as repressor. PHOX2B-mutant represses DBH, while PHOX2B-wt activates DBH (no noradrenalin, only dopamin??).
- Mutation ALK:
is a RTK. ALK innhibition induces tumor regression (therapeutic inhibitor?)
- High stage tumors have partial chromosomal gains/losses (low stage have whole gains/losses).

What are the most frequent mutations in neuroblastoma?

- ALK, ATRX, ODZ3, PTPRD (found in 5 tumors)

* Found by sequencing 110 neuroblastoma by whole genome, paired-end sequencing
* Number of mutations correlated with stages

What is the hallmark of pediatric tumors?

Very few mutations in childhood tumors

What are the consequences of trisomies?

- Trisomy 21 = down syndrome
- Trisomy 13,14 = very severe symptoms
- Any other trisomy = prenatal death

What are the consequences of a deletion (loss of 1 copy)?

- The loss of 1 copy leads to a 30-50% reduction in expression of hundreds of genes

What role do the adherens play in EMT?

- Adherens junctions keep the epithelial cells together
- Adherens junction: E-cadherin, catenins, actin

- In EMT the E-cadherin is down-regulated --> loss off adherens junctions --> cell migration + ability to down regulate other cells.

When and when does EMT occur in normal development?

- Mesoderm EMT (primitive streak):
Cardiac cells, Hematopoitic cells, Urogenital tract, Embryonic notochord, Bones, Skeletal muscle and tendous, Digestive tract (endoderm)

- Neural crest EMT:
Neurons & glial cells, melanocytes, cranial bones & muscles, odontoblasts

What are pathogenic forms of EMT?

Fibrosis, tumor progression

What are the characteristics of EMT and MET?

EMT
- Activation EMT-TFs
- E-cadherin repression
- Loss apico-basal polarity
- Invasion & migration
- Low proliferation
- Intravasation & extravasation (tumor cells)
- Survival
- Chemotherapy resistance
- Inflammation

MET
- Repression of EMT-TFs
- E-cadherin transcription
- Apico-basal polarity
- Cell-cell adhesion
- Increased proliferation

For what is EMT (normally) and MET?

- EMT: embryonic cell migration, wound healing
(cancer cell dissemination)
- MET: organ differentiation, epithelial homeostasis
(metastatic colonization)

What are the (main) EMT transcription factors?

EMT transcription factors = Snail, Slug, twist, Goosecoid, FOXC2, ZEB1, ZEB2

What are epithelial and mesenchymal markers?

- Epithelial markers: E-cadherin, alpha-catenin, Beta-catenin, gamma-catenin
- Mesenchymal markers: fibronectin, vimentin, N-cadherin, alpha-SMA

What is the E- to N-cadherin switch in EMT?

- With E-cadherin the cell is in epithelial state and interacts with the melanocytes with the help of gap junctions
- With N-cadherin the mesenchymal cells can interact with other mesenchymal cells

What is the invasion-metastasis cascade?

Primary tumor formation --> Localized invasion --> Intravasation --> Transport through circulation --> Arrest in micro-vessels various organs --> Extravasation --> Formation micrometastasis --> Colonization: formation macrometastasis

* Intravasation in lymphe & bloodstream
* Environment very harsh to survive + high physical stress

How is EMT induced?

EMT is induced through concerted actions of paracrine and juxtacrine signals.

- Paracrine: MSCs, fibroblasts & myofibroblasts
- Juxtacrine: Macrophages

Which 2 cell types are there in neuroblastoma?

Mesenchymal
- undifferentiated
- Migration-high
- Neural-crest like
Adrenergic
- Lineage commited
(adrenal precursors)

* The 2 types are genetically identical & can transdifferentiate --> epigenetic mechanism

How does chip-seq work?

You can use chip-seq to recruit chromatin modifications: antibody recognizes H3K27ac --> recruits only this chromatin --> remove DNA from histone --> enrich only DNA with H3K27ac.

Peak: enrichment modification (H3K27ac)
--> map modification on genome

Why is intra-tumor heterogeneity important clinically?

The primary tumor gets treated with the first-line treatment. This results in a residual disease with variable fitness. However, when this relapses there will be a change in phenotype: the cells that survived the previous treatment will now thrive. The second-line treatment won't work and will result in treatment failure because it is the same treatment as used on the primary tumor.

Of which cells does the primary tumor mainly consist of?

Primary tumor consist of ADRN-type cells
- Minor population MES cells

MAML3/PRRX1 double staining: neuroblastoma PRRX1+
--> not related to stage, 2% PRRX1+ primary tumors

Which cells in neuroblastoma are therapy-resistant?

- MES cells are drug-therapy (chemo) resistant, they survive high concentrations of chemo while ADRN cells die.
- Many post-treatment tumors shift expression towards MES stage (however, there are some which shift into 'super-ADRN' state)
So, after chemo --> enrichment MES
- Retinoic acid: induces differentiation, stops proliferation. While ADRN cells respond and go into apoptoses, the MES cells do not respond.
--> MES cells resistant to chemo & retinoic acid
- Relapse: tumor has a strong PRRX1 expression (this is inversely correlated with primary tumor, reduction ADRN, increase MES).

What is MRD-monitoring? And what is the current problem with it?

MRD: minimal residual disease
- Measures response to therapy
- Clear bone marrow & blood from neuro blastoma
- At diagnosis, chemo, after surgery and after retinoic acid

- MRD-monitorting is based on ADRN mRNA markers: PHOX2A, PHOX2B, TH, DDC, DBH.
- The ADRN cells are sensitive to therapy, but the ones that escape are the MES cells, which are missed by the MRD.

* Tumor-specific DNA translocations provide alternative MRD-mark!

What would a successful therapy look like for neuroblastoma?

- It will most likely eliminate both the MES and ADRN cell types.

* combination BMI1 and EZH2 --> hardly any tumor formation

How does the embryonic development of the ICM go?

ICM = inner cell mass
- Blastocyst contain the inner cell mass, a cavity and trophectoderm
- The inner cell mass (ICM) contains embryonic stem cells, they have the capacity to generate all lineages in the embryo (all 3 germ layers).
- Epiblast: generates embryo
- Primitive endoderm & trophectoderm: contribute to extra-embryonic tissue

What are the downsides of ES therapy?

- Immune system 'rejects' strange cells
- Ethical arguments not to use human embryos for experiments

How can cells be reprogrammed back to pluripotency?

1. Nuclear reprogramming
Take out nucleus from oocyte, put nucleus somatic cell in (transplant) --> blastocyst --> ES cell.
- Somatic cells can be 'reprogrammed' to embryonic state

2. Transcription-factor induced reprogramming
Somatic cell + c-Myc, Oct4, Sox2, Klf4 --> iPS cell
- Patient-specific cell therapy, drug screening, human disease models
- Not rejected by immune system (as it is derived from own fibroblast)

What are the TFs to induce iPS cells?

OSKM set iPS: OCT3/4, SOX2, KLF4, MYC

How does chromatin structure affect cellular identity and state transitions?

- Normal: cell state changes accompanied by epigenetic state changes
- Restrictive: Epigenetic insult raises chromatin barrier. Cell unable to leave proliferative state.
- Permissive: Epigenetic lesions lower chromatin barrier. Plasticity allows bidirectional transition.

What is direct reprogramming?

For direct reprogramming there is no ES cell/iPS intermediate required.

What is the worklflow of single cell RNA sequencing?

Single cell --> RNA --> cDNA --> amplification cDNA --> single cell expression profiles --> cell type identifications .

- captures transcriptional information (mRNA)

What shapes the unique identity of cancer cells?

- Environmental stimuli
- Cell development
- Cell cycle
- Spatial context

What are the applications for single-cell RNAseq?

- Deconvolving heterogenous cell populations
- Trajectory analysis cell state transitions
- Network interference
--> identification different cell types

How does the developmental hierarchy of oligodendroglioma cells differ?

- Stem-like, AC-like and OC-like state can be separated by single-cell Seq --> plotted with stemness score & lineage score (developmental hierarchy)
- The pattern is basically the same, but the distribution (percentages) is skewed across tumors.
- All different stages are found, but distribution differs

Which cells are enriched among stem/[progenitor cells?

Cycling cells are enriched among stem/progenitor cells --> stem/progenitor cells cycle actively (proliferate actively)

What is ChIP-seq? How does it work?

- Chromatin Immuno Precipitation and Sequencing
- Measures modifications of chromatin along the entire genome
- Identifies transcription factor binding sites along the entire genome

There is a specific antibody for specific modification OR for specific transcription factor --> only sequencing DNA with those specific modifications.

What are the possible epiginetic modifications of chromatin?

- Methylation, phosphorylation, acetylation, ubiquitylation

* Euchromatin = open
* Heterochromatin = packed

How did iPSC cells reveal the existence of super enhancers?

- iPSC have small group of highly expressed transcription factors (Oct4, Sox2, Nanog, Klf4)
- ChIP-seq for these transcription factors shows that they all strongly bind to a few specific regions in the genome
- These regions also bind typical enhancer proteins, like Mediator complex, Cohesin and CTCF.
- These regions are called super enhancers.

How does a Core Regulatory Circuitry (CRC) work?

1. TFs bind to their own enhancer
2. TFs bind to one another's enhancer
3. TFs form feed forward network
4. TFs program lineage identity

How are super enhancers marked?

- Very strong H3K27Ac, but abscence H3K4Me3
- Strong TF binding signals

How do CRCs function as the central control system of cell type switch?

MES CRC transcription factor can impose a switch in phenotype (ADRN--> MES) associated with switch in super enhancers to MES-type.

- Some CRC transcription factors are mutated in neuroblastoma

How does RTK signaling work?

Signal molecules (like growth factors) bind to the RTK which autophoshorylates. The adapter complex then can bind to the RTK and transform Ras-GDP into Ras-GTP (active form). After this the RAS-RAF-MEK-ERK cascade follows.

* RAF requires dimerization to signal
* ERK induces proliferation
* ERK induces negative feedback (DUSP - selfinhibition & inhibition RAF).

So:
1. Ligand binds & activates RTK
2. RTK activates RAS-RAF-MEK-ERK
3. ERK induces transcription

What happens if there is a mutation in Ras?

- RAS is the most frequent oncogenic mutation in cancer
- When RAS is mutated it no longer depends on the binding of the ligand (growth-factor), which causes uncontrolled growth

Mutation: strong activation & reduced inhibition (cause it does not respond to it anymore)
--> hyper-activation RAS-RAF pathway

How does a mutation in BRAF induce cancer? How does it affect the RAS-RAF-MEK-ERK cascade?

- BRAF is a high frequency mutation in cancer
- Mutations (hotspots) in:

Activation loop (A-loop)
Phosphate-binding loop (P-loop) of kinase domain

- Normal: A & P loops are interacting --> protein is inactive
- Mutation: disturb this inactive conformation --> activation protein (permanent signaling to downstream kinases) --> hyper-activation downstream pathway

* Mutant BRAF shows strong kinase activity

RAS-RAF-MEK-ERK
BRAF mutation --> RAF mutated & hyperactive --> higher levels phospho MEK & ERK --> higher level downstream TFs
* Negative feedback is also reduced!

Why are there 2 cell types in cancer/neuroblastoma?

- Dedifferentiation is the 'wrong' direction of development. This suggests that ADRN cells do not fixate their cell state in the right way.

- Cell state is fixated by:

Core regulatory Circuitry (CRC)
Epigenetic modifiers/chromatin modifiers

How is the epigenetic switching between 2 cell types regulated in neuroblastoma?

MES --> ADRN:
Suppression by polycomb genes
Activation by trithorax genes

How do the polycomb genes work?

- Polycomb genes = epigenetic suppressors

PRC1 complex, PRC2 complex

- Step 1: PRC2 methylates H3K27 --> H3K27me3

H3K27me3 is a silencing mark
H3K27me3 is added both to (super)enhancers & gene starts
Replaces the activation mark H3K27ac

Core components PRC2 = EZH1, EZH2 (methyltransferases), SUZ12, EED, RBBP

Mutations: in EZH2, the methyltransferase. Mutated EZH2 represses genes for cell cycle exit.

- Step 2: PRC1 recognizes & binds to H3K27me3 mark placed by PRC2 --> adds ubiquitin mark to H2AK119 --> strong compaction chromatin --> closed to transcription

How do Trithorax genes work?

- Thrithorax genes = epigenetic activators (chromatin remodelers)

COMPASS complex, ASHI/CBP comples, SWI/SNG (BAF) comples

- Default state of genome is silenced. Chromatin remodelers can open specific regions by removing/displacing nucleosomes.

Can PRC1/2 control transdifferentiation?

PRC1 in haematopoietic stem cells:
- Mainly CBX7 expressed
- CBX7 overexpression enhances self-renewal & proliferation
- Overexpression CBX2,4,8 results in differentiation
- CBX7 and CBX8 bind to partly overlapping genes

CBX7 represses differentiation genes
CBX8 represses stem cell genes

Neuroblastoma

72 important questions on Neuroblastoma