Regulation of hematopoietic differentiation Flashcards
What 4 broad steps are included in differentiation?
- Lineage commitment
- Cell proliferation
- Cell survival
- Differentiation
How many RBCs are produced per day? WBCs? Platelets?
o ~ 200 billion erythrocytes/day
o ~ 100 billion leukocytes/day
o ~ 100 billion platelets/day
What is a growth factor?
Growth factor = protein that promotes the proliferation of cells
Who first discovered EPO, and when?
o Early 20th century: hémopoïétine (Paul Carnot)
Experiment with rabbits
Name 2 important people in the identification of GFs in hematopoiesis
Don Metcalf and Richard Stanley
What is EPO?
Erythropoietin (EPO) is a peptide hormone (GROWTH FACTOR) that is produced naturally by the human body. EPO is released from the kidneys and acts on the bone marrow to stimulate red blood cell production.
What does EPO do on cell lineage?
Favorizes differentiation of Megakaryocyte-Erythroid precursord to Erythroid precursors (EP) rather than MkPand favorises EP differentiation to erythrocytes
What growth factor is useful for differentiation of MEP to MkP and MkP to platelets?
TPO (thrombopoietin): plays a big role in megakaryocyte development and stabling factor
What is the main category of growth factors acting on the lymphoid lineage?
IL (Interleukins)
What growth factor favorizes the differentiation of GMP to Monocytes?
M-CSF
What growth factor favorizes the differentiation of GMP to Neutrophils and eosinophils?
G-CSF, IL-5, SCF
What is a colony-forming assay?
Introduced around 50-60 years ago
- Limited number of bone marrow (or fetal liver) cells are plated on semi-solid media containing growth factors –> proliferation, differentiation of hematopoietic progenitor populations - growth of colonies (1-2 weeks) and assessment of number of colonies, size and morphology
Isolate cells –> dilution of cells in medium –> plating into semisolid medium (methylcellulose) –> incubate (colony identification depending on growth factors given)
Name and describe the possible colonies on the Myeloid lineage of a colony-forming assay
CFU-GEMM (CFU-Granulocyte, Erythrocyte, Monocyte, Megakaryocyte)
- -> CFU-Mk
- -> BFU-E (Burst-forming unit erythrocytes) –> CFU-E
- -> CFU-GM (Granulocyte, monocytes) –> Either CFU-G or CFU-M
CFU-GEMM – couple thousand cells
BFU-E – More compact, couple hundred cells
CFU-E – more compact and restricted in lineage (about 50 cells)
Name functions of the granulocyte-colony stimulating factor (G-CSF)
- Functional stimulation
- Maturation induction
- Differentiation commitment
- Proliferation
- Survival
- -> makes the cells becoming a macrophage, neutrophil etc.
What is KIT?
A growth factor in the form of a receptor
What is the pathway used by KIT to relay signal?
It has tyrosine kinase activity
KIT is present in how many % of bone marrow cells? of CD34+ cells?
- Present in 1-4% of bone marrow cells
- Present on 60-75% of CD34+ cells
What are the domains of the KIT receptor?
- Extracellular domain
o Made of immunoglobulin-like domains
D1-3: ligand binding (binds SCF)
D4-5: dimerization (activation of receptor) - Transmembrane domain
- Juxtamembrane domain
o Includes tyrosine kinase domains
What are other names given to the KIT receptor?
c-kit, CD117
What are the different names for the ligand binding to KIT?
KITLG (kit ligand) (other names: SCF (stem cell factor), steel factor)
What is the GNNK domain?
A domain on the transmembrane part of the KIT receptor
Glycine, asparagine, Lysine
The two major splice variants of KIT differ by the presence or absence of four amino acids (GNNK) at the juxta-membrane region of the extracellular domain
What are the differences between KIT GNNK- and GNNK+
GNNK-
- Anchorage independant growth
- Loss of contact inhibition (focus formation)
- Tumorigenicity
- More rapid and extensive tyrosine auto-phosphorylation and rapid internalization
GNNK+
- Colony formation but poor focus formation
- No tumorigenicity
How are KIT receptors activated?
Homodimer of SCFs (ligands) brings receptors together
Ig domain 4 and 5 (D4-5) make the connection – dimerization –> phosphorylation of intracellular domain
Which pathways are initiated when KIT receptor is activated by its ligands? What are the different pathway’s functions?
- Mitogen-activated protein kinases –> proliferation
- AKT kinase –> survival
- PKC –> regulation of gene expression (e.g. activations of TFs, etc.)
How is KIT negatively regulared?
negative regulation (termination of signalling) via phosphatases such as SHP-1, SHP-2 and SHIP
bonus
bonus
What does a mutation in the ligand binding domain of KIT cause as a disease?
idiopathic myelofibrosis
What does a mutation in the extracellular Ig domain of KIT cause as a disease?
AML
What does a mutation in the transmembrane domain of KIT cause as a disease?
acute leukemia
What does a mutation in the juxtamembrane domain of KIT cause as a disease?
gastrointestinal stromal tumors (GIST)
What does a mutation in the phosphotransferase domain of KIT cause as a disease?
mastocytosis, mast cell leukemia
What are the most common types of mutations in the KIT receptor?
Intracellular
What is the gleevec/imatinib drug? what is its function?
- originally developed for inhibition of BCR-ABL oncogenic fusion protein
- inhibitor of enzymatic (tyrosine kinase) activity of KIT; treatment of chronic myeloid leukemia, GIST
- mutations of enzymatic site confer resistance to gleevec/imatinib (resistance to drug)
Name and explain the two models for lineage commitment in hematopoiesis
Instructive model (inductive)
a. Growth factors transmit specific signals to stem cells or multipotential hematopoietic cells, directing their lineage commitment and differentiation.
b. Do they make a specific cell commit to a specific lineage?
Permissive (stochastic) model
a. Lineage commitment and terminal determination are intrinsically determined with cytokines (growth factors) providing only permissive growth and survival signals.
b. No role in cell commitment to lineage but has role in growth and survival?
Explain Till and McCulloch’s experiment and which model of lineage commitment it favours.
Mouse spleen colonized by CFUS; after 10 days, different colonies present in the spleen (not all same size)
Frequency and size of colonies best fitted by random distribution (some differentiated right away while others take more time) –> large colonies are the ones that differentiated first
“birth” (self-renewal) and “death” (differentiation) occur as probabilistic (stochastic, permissive) events –> Favors permissive (stochastic) process: decision between A cell becoming 2 A cells or A and B cells is random
How does the EPO receptor work? What happens to mice are KO for EPO gene?
Similar to KIT, also functions as a dimer
KO mice studies have been done; KO of EPO (gene E13) very pale embryo, died shortly after 14 days of gestation (vs. 20 days needed to give birth)
Wildtype had a redish liver. None in KO mice
Cells from fetal liver:
- Wildtype has many different cells that represent many stages of erythroid cell synthesis, eventually lose their nucleus
- KO: Still makes proerythroblasts, thus can see cells have commited to erythroid lineage but do nut mature as RBCs.
- Wildtype: more # of nucleated cells in liver as multiple developmental stages
- KO low number.
- More CFU-E and BFU-E in KO mice as compared to wildtype. These cells are blocked at the CFU and BFU stage –> Epo knock out animals have committed erythroid progenitors (CFU-E, BFU-E), but no mature erythroid cells
What experiment supports the permissive role for lineage commitment?
EPO KO mice
Epo knock out animals have committed erythroid progenitors (CFU-E, BFU-E), but no mature erythroid cells
Does CFU-GEMM need EPO to differentiate to an early RBC (BFU-E)?
No but cant mature well without it
If GMPs are cultured with M-CSF, what will you get?
macrophages and monocytes
If GMPs are cultured with G-CSF, what will you get?
Granulocytes
What role does M-CSF and G-CSF have in lineage commitment?
Instructive role
Name 3 pieces of evidence that show no/minor role in lineage commitment.
- presence of progenitors in knock out mice (e.g. EPO, EPOR)
- lack of specificity in signaling by cytokine receptor (e.g. prolactin receptor can replace EPOR)
Name 3 pieces of evidence that show role in lineage commitment.
- bioimaging/long-term observation studies showed that cytokines (M-CSF, G-CSF) can instruct hematopoietic lineage choice
- expression of exogenous IL-2bR and GM-CSFR in common lymphoid progenitors, and treatment with either IL-2 or GM-CSF, leads to reprogramming into granulocytes & macrophages, respectively (does not work for IL-7R or EPOR)
- Recent evidence that some cytokines can transduce a genuine lineage-determining signal
What is a transcription factor?
Protein that binds to DNA and controls the rate of transcription
Name TFs involved in the specification of HSC
SCL
LMO2
Name TFs involved in the expansion/maintenance of HSC
GATA-2
AML1
Name a TF involved in the commitment to lymphoid lineage
Ikaros
Name a TF involved in the commitment to MEP lineage
C-MYB
Name a TF involved in the commitment to RBCs
GATA-1, EKLF
Name a TF involved in the commitment to neutrophils
C/EBPalpha
What is Ikaros? What happens if KO of ikaros?
- Functions as a tumor suppressor
- Inhibition of Ikaros activity leads to leukemia
- Phosphorylation by CK2 at multiple sites –> blocks function and increases degradation of Ikaros
- Inhibits PI3K pathway and cell cycle (growth)
- CK2 phosphorylates Ikaros = More cell cycle and PI3K pathway–>leukemia progression
- Block CK2 with CK2 inhibitors (CX-4945) –> Leukemia inhibition
Loss of function of Ikaros is linked to many cancers, especially leukemias
What drug can be used to inhibit leukemia? How do they work?
CK2 inhibitors
Inhibit CK2 which prevents CK2 from phosphorylating Ikaros. Thus Ikaros stays on the DNA and prevents transcription of genes for cell cycle and PI3K pathway, inhibiting them
What happens if too much CK2?
Ikaros is phosphorylated and unbinds from DNA. This increases cell cycle and PI3K pathway –> leukemia
What is a zinc finger? Name an example of protein that has a zinc finger domain
Zinc finger domain important for DNA binding or interaction with other proteins
- promotes differentiation
- inhibits proliferation
- controls migration and adhesion
- regulates gene expression by chromatin remodeling
Ikaros is an example
What happends to people who have an ikaros gene deletion?
- Ikaros deletions: o 80% of BCR-ABL1-positive ALL patients; o 5% of patients with T cell ALL KO mice for Ikaros much smaller mice; misses thymus organ thus cannot make T-cells or B-Cells No cells positive for CD4 and CD8
What is C/EBPalpha?
CCAAT/enhancer-binding protein alpha
Favorizes lineage commitment to granulocytes and monocytes.
What happens to people/mice with KO of C/EBP alpha?
less monocytes (about half) and no granulocytes
What is NF-E2?
basic-leucine zipper (bZIP) transcription factor
heterodimer (two subunits: p45 NF-E2 & a small Maf protein)
large subunit specifically expressed in erythroid, megakaryocytic and mast cell lineages
The basic domain confers DNA binding and the neucine zipper mediates dimerization
What happens to NF-E2 KO mice?
KO mice are smaller, paler, have internal hemorrhages and die shortly after birth
Phenotype in in megakaryocytes (precursor for platelets)
They do make megakaryocytes but no platelets (they don’t shed…) –> thrombocytopenia
Explain the cross-antagonistic transcription factor model
In uncommitted cells (progenitor cells), there are TFs A and B in about equal amounts (cancel each other out)
A stochastic events leads to an increase in one of the two TFs.
- More A –> A binds target gene and then cell becomes an A cell
- Same for B
Balance between transcription factors will dictate differentiation
Not only about TFs but also cofactors and others.
Explain the cross-antagonism between C/EBP and FOG
Under normal circumstances and controlled conditions, transcriptional inhibition of FOG by C/EBP TF –> commitment to eosinophil lineage
Artificial overexpression of FOG –> FOG interacts with GATA1 which will block the transcription of other proteins important for eosinophil lineage (negative regulation)
–> reverse back to precursor state
What is FOG?
Friend of GATA1
Which GATA is mostly present in the early stages of erythropoiesis? Late stage? How does it happen?
GATA2 early
GATA1 late
GATA2 drives its own expression –> very active at early stages
When GATA1 expression rises, it interracts with NuRD (Nucleosome remodeling and deacetylase) and FOG-1 and this complex binds to the same sequence (WGATAR sequence) and blocks the expression of GATA2
Name 3 TFs involved in leukemia chromosomal translocations
Ikaros, SCL, LMO2
Name 2 TFs identified in Gene KO studies
Ikaros, NF-E2
Name 3 TFs that are critical for lineage commitment which was found in gene KO studies
SCL, Ikaros, GATA-1
Name 2 TFs that have a cross-antagonism model
C/EBP and FOG
Name TFs essential for cellular reprogramming (iPSCs)
Oct3/4, Sox2, c-Myc, KLF4
What are KIT’s roles in HSC and early hematopoietic cells?
o proliferation and survival (in synergy with other growth factors)
What are KIT’s roles in mast cells and dendritic cells?
o fully differentiated cells maintain high levels of c-Kit expression
o proliferation and survival function
What are KIT’s roles in erythroid cells?
o promoter of erythroid colony formation but no total dependency on KITLG (SCF) for erythropoiesis
Name other roles of KIT
Other roles in…
- Pigmentation – proliferation, survival and migration of melanocytes
- Reproduction – protection of germ cells
- Gastrointestinal tract – motility
- Nervous system – spatial learning
- Cardiovascular system – cardiac stem cell and cardiomyocyte terminal differentiation
- Lung – maintaining lung tissue integrity
