JAK/STAT signalling

Question 1

Give a simple outline of the JAK/STAT signalling pathway?

Answer 1

1) TMD receptors associated with the ICD of the Jak kinase are latent in the membrane
2) Binding of the ligand, causes dimerisation and activation via tyrosine phosphorylation
3) Phospho-y in correct context recruits cytosolic STATs
4) STATs are activated by tyrosine phosphorylation of themselves and dimerise
5) STAT dimers translocate to the nucleus, bind to DNA and activate transcription

Question 2

What are the main roles of JAK/STAT signalling?

Answer 2

Humans have lots of ligands and receptors that use JAK/STAT signalling. Four JAKs: 1, 2, 3, type2
Seven STATs: 1, 2, 3, 4, 5a, 5b, 6
STATs 3, 4, 5, 6 all heavily involved in the differentiation of T helper cells.
-Anti-inflammatory cytokines such as IL4, IL5, IL10 and TGFb [Th2 cells]
-Pro-inflammatory cytokines such as INFy, TNF-a, IL2, IL6, IL12 [Th1 cells]
These signals use JAK/STAT signalling.

Question 3

How is JAK/STAT involved with haematopoesis?

Answer 3

Haematopoietic stem cells reside in the bone marrow. The process of maintaining and differentiating these stem cells is orchastrated by ligands via JAK/STAT pathway ligands like IL3, thrombopoietin, erythropoitin and GM-CSF. Gives rise to platelets, erthrocytes, monocytes as well as granulocytes (eosinophils, neutrophils and basophils).

Question 4

What happens when JAK2 is knocked out or has a gain of function mutation?

Answer 4

JAK2 KO is embryonic lethal because there is an absense of erythropoiesis. Gain of function causes Polycythrmia vera (PV), Primary Myelofibrosis (PMF) and Essential thrombocythaemia (ET).

Question 5

Why are STAT5a and 5b important?

Answer 5

STAT5a and 5b are important because they are mediators of TPo and EPo signalling ligands such as IL2, EPO, TPO, GM-CSF and IL7.

Question 6

What is EPOR, TPOR, GM-CSFR?

Answer 6

EPOR is the receptor for Erythropoietin. Absence is embryonic lethal. EPO is used as an endurance drug to give more RBC, more O2 to muscles. TPOR or C-MPL is a receptor for thrombopoietin, KO has reduced Haemopoitic

Question 7

What is JAK/STAT component crosstalk?

Answer 7

Antibodies specific to STATs or phosphotyrosines show that there is a degree of crosstalk between ligands and others can stimulate other STATs. There are differences in stimulation depending on the ligand. IL6 can activate STAT3 and a little bit of STAT1. The vertebrate JAK/STAT pathways are complex, they have compensatory mechanims AND crosstalk.

Question 8

What is the IL6-like family?

Answer 8

Parallel alpha helices (up-down-up-down topology) in ligand with a few binding loops. Crossover of different ligands doing the same role. Examples: IL6, IL11, LIF (leukaemia inhibitory factor), OSM (Oncostatin M), Ciliary neurotrophic factor, cardiotrophin-1, cardiotrophin-like cytokine.
Activate target genes in differentiation, survival, apoptosis and proliferation. Bind to PM complexes containing gp130.

Question 9

What are the two types of IL-6 family receptors?

Answer 9

The receptors are heterodimeric glycoproteins combined with different things.

• Some have gp130 with a non-signalling a-receptor which has ligand binding sites but nothing on ICD e.g. IL6Ra, IL11Ra
• Others have gp130 with a full length signalling B-receptor which has a JAK/STAT binding site
  e. g. OSMRB, LIFRB

Question 10

What are the interactions between IL6 and its receptor?

Answer 10

Site I binds with a shorter a-receptor, Site II and III bind with longer B receptor. Cytokines interact with the receptor via structurally defined sites. Ligand and receptor must be close because ligand is at such a low concentration. Following ligand binding, receptor/ligand complexes are rapidly internalised via cytoplasmid dileutice motif in B receptors. Ligand-dependent receptor activation and signal transduction depends on the 3 membrane proximal FNII domains. On the intracellular side, there are BOX I, II, III domains JAK kinase binds to conserved BOX I and II but BOX III is less common.

Question 11

What does the drosophila JAK/STAT pathway look like?

Answer 11

Three ligands: Upd, Upd2, Upd3
Two receptors: Domeless and Latran
One JAK: Hopscotch
One STAT: STAT92E

If there is a mutation in JAK or STAT there is a complete block of all signalling. The phenotypes of -/- Upd, Dome and STAT are very similar. Missing segments, loss of posterior features and messed up head structures.

Question 12

How is dimerisation of domeless receptor visualised?

Answer 12

Fused Domeless receptor with deletions in B-galactosidase to give mutated fusion proteins where only one half is active. Use a bi-molecular complementation assay. You will only get a functional B-galactosidase enzyme when variants are very close together (dimerisation), only producing blue colour in specific tissues. Localised B-galactosidase activity and therefore proteins are expressed everywhere but they are only dimerised in very specific tissue e.g. hindgut, posterials sphericals. Regulated spatially and temporally.

Precautin should be taken to use different inducing lines as unspecific dimerisation can be observed with unusually high levels of hybrid protein expression.

Question 13

What can hemapoietic stem cells differentiate into?

Answer 13

Stem cells in blood can differentiate into:

• Plasmocytes: macrophages that clean up cell debris, bacteria and fungi
• Crystal cells: involved in melanysation response (wound healing, clotting)
• Lamellocytes: large cells that engulf the eggs of parasitic wasps

Question 14

How are drosophila blood cells similar and different to ours?

Answer 14

Drosophila have circulating blood cells that fight infection and promote clotting in the event of an injury.

BUT… Don’t have blood cells that carry oxygen because flies transport oxygen via trachea. Meaning insects can’t grow over a certain size.

Question 15

What are the two rounds of haemopoiesis?

Answer 15

-Embryonic
Population of haemocytes orginates in the precephalic mesoderm, migrate and disperse by lamellipodia throughout the embryo along programmed pathways. 95% plasmocytes, 5% crystal cells that don’t migrate.
-Larval
Occurs in lymph glands located either side of the heart. Contains primary, secondary, tertiary lobes. In primary lobes there are two regions (inner medullary zone and outer cortical zone). These contain haemocytes which can be differentiated by various expressions of genes including domeless and Upd3. Posterior signalling centre releases these signalling molecules. Stem cells differentiate and get pushed out. Gland bursts and cells released into bloodstream. Clear up apoptosis due to metamorphosis. Adult doesn’t make any new blood cells.

Question 16

How do wasp parasites affect the drosophila larva?

Answer 16

Wasp parasites lay their eggs in late L2-late L3 drosophila larvae. Unfertilised eggs become males as they’re haploid. Eggs compete in th elarva and only one survives. Surviving egg will eat larvae alive and develop as a pupa within the husk of the larva. It will hatch from their hosts 4-5 weeks later. In the presence of a wasp infection, haemopoitic progenitors will differentiate to produce lamellocytes which can encapsulate egg to protect against the parasite.

Question 17

What is the hop tuml drosophila mutation?

Answer 17

Gain of function, temperature-sensitive JAK mutation. Toll usually degrades factor inhbiting NFkB transcription but overexpression of the NFkB transcription factor Dorsal in larvae leads to lamellocyte differentiation.
Extra lamellocytes which end up attacking each other creating a lethal tumour. In a WT fly, there are 1.5 x 10^6 cells per ml, 5-6% of which are lamellocytes. In hop tuml, the amount changes depending on the temperature. So in low temp, there are more blood cells at 17C but at higher temp, there are fewer cells but a greater percentage are lamellocytes. The larva wrongly thought it was being infected with a wasp.

Same occurs with gain of function mutations in humans.

Question 18

How are EPO and TPO different?

Answer 18

TPO and EPO both cause differentiation but have extracellularly diffusible proteins that bind to their own receptors.

TPO-MPL-JAK2-STAT3/5a/5b-Target genes
EPO-EPOR-JAK2-STAT3/5a/5b-Target genes

Question 19

What are the types of myeloproliferative neoplasms?

Answer 19

Primary myelofibrosis
Men and women over 60. Bone marrow function is impacted by scarring. Associated symptoms and enlarged spleen. Can be found as primary MF or as a progression.

Essential Thrombocythemia
Women over 50. Blood malignancy due to elevation of platelets in the blood. Common symptoms include blood clotting and bleeding. Risk of progression to MF.

Polycythemia Vera
Men over 60. Elevation of red blood cells, white blood cells, platelet counts as well as an enlarged spleen. Thicker denser blood smear.

Question 20

What is the structure of JAK2?

Answer 20

Various domains at the N terminus for protein-protein interactions (SH2-like domains). JH1 is the kinase domain that phosphorylates STAT. JH2 is thought to be a pseudo-kinase domain, still a kinase but neg regulates JH1.

Question 21

How is JAK2 linked to myoproliferative neoplasms?

Answer 21

If you mutate residue in JH2, negative regulation is reduced, so activity of JH1 is increased.
In PV, >98% of patients have JAK2 mutation.
In ET, 50-60% of patients have JAK2 mutation.
In PMF, 50-60% of patients have JAK2 mutation.

Question 22

How did a mutation in JH2 domain lead to increased JAK/STAT pathway activity?

Answer 22

JAK2 has a similarity to Hopscotch in drosophila which also causes overproliferation. Luciferase ratios showing the activity of JAK/STAT signalling pathway uses the ability of STAT to turn on a gene that makes luciferase. Adding JAK2 mutant (which contains gain of function mutation in JH2 V617F valine to phenylalanine [acquired, not hereditary, may be caused by duplication in mitoitic recombination]), the basal activity increases. This mutation inhibits the movement of the activation loop from its inactive to active conformation. If EPO receptor is transfected, the activity increases further. JAK2 mutant still able to respond to ligand.
Average survival is 9.2 years after diagnosis.

Question 23

What effect does mutations in MPL have on the activity of the JAK/STAT pathway?

Answer 23

MPL is normally presented as a monomer with a JAK attached. The ligand will bring 2 monomers along to initiate activation. However, in the gain-of-function mutation in the TMD causes the 2 monomers come together in the absence of ligand and activate the downstream pathway. To mirror the mutation, cysteine is added to cause sulfide bridges to form. This is sufficient to activate phosphorylation.
This MPLW515L mutation is a somatic mutation and is not a SNP. Hypersensitive to TPO giving a selective proliferative advantage.
Average survival is 9.1 years after diagnosis.

Question 24

What effect does mutations in Calreticulin (CalR) have on the activity of the JAK/STAT pathway?

Answer 24

Calreticulin is a chaperone that resides in the ER. A frameshift mutation (caused by insertion or deletion) that replaces the C-terminus of WT CalR with a common novel peptide sequence. The mutation may be different further up but all C-termini have this same replacement. Both the losing and the gaining of sequence contribute to the mutation. Likely that this impairs the calcium-binding activity of calreticulin.
In CalR there is growth in the absence of the ligand. As you increase the level of ligand the mutant grows even faster than WT.
A 52-bp deletion and a 5-bp deletion are found in more than 80% of CALR-mutant patients.
Average survival 17.7 years after diagnosis.

Question 25

What did screening for JAK/STAT drugs elucidate?

Answer 25

A library of 2000 FDA-approved drugs, agrochemicals, cytotoxiz chemicals and pure natural products were screened for their activitiy in the JAK/STAT pathway. Used a high throughput transcriptional reporter assay. Sorted the components by their effect. Methotrexate and Aminopterin stood out, similar in their effect to decrease phosphorylation of STAT5 in a dose-dependent manner.

Question 26

How was methotrexate tested in mouse models?

Answer 26

Cohorts of WT, heterozygous and homozygous hJAK2 V617F siblings. Human JAK2 recombinantly knocked into JAK2 locus, protein made is the mutant hJAK2. All mice have same genetic background. Treated with 5mg/kg MTX by IP injection every 2/3 days for 28 days (dose for RA). PBS or ruxolitnib used as control. Treated for 6-7 weeks and then analysed at 10/11 weeks of age. Protein homogenate taken from the spleen. Only pSTAT found in the homozygous mice, when treated, increased pSTAT abolished. After treatment, levels of PIM1, a JAK/STAT target gene is also decreased back down to normal levels. Decreased size of spleen combatting secondary haematopoiesis. Significant difference in haemoglobin and WBC levels.

Question 27

Give two case studies of how Methotrexate has been used to treat myoproliferative neoplasm patients?

Answer 27

57 yo femaile ET patient with scleroderma. Intolerant to current treatment. 10mg/week of MTX normalised platelet count within 45 days.

69 yo male PV patient with RA. Difficult venous access, intolerant to treatment. 10mg/week MTX, Hematocrit (5 of RBC) reduced, splenomegaly and pruritus (itching) in remission.

Question 28

What is Methotrexate?

Answer 28

Really old drug (40/50s)

• Low dose used to inflammatory and autoimmune diseases
• High dose for chemotherapy

First-line treatment for RA, effective, safe, well-tolerated. Unknown mechanism. Efficacy for RA is likely due to suppression of JAK/STAT pathway, as it is key for inflammatory progression.

Question 29

How do Ruxolitinib and Methotrexate compare for treating Myoproliferative neoplasms?

Answer 29

Ruxolitinib is a JAK/STAT pathway inhibitor and effective for MPNs but it has not been approved by NICE on cost effectiveness grounds.
£43,200 pa (only approved for late-stage MF).
MTX seems to act via the suppression of JAK/STAT mechanism, so could be used to treat MPNs for £32 pa.
Clinical trials underway soon.

Question 30

What are plasmatocytes?

Answer 30

Professional phagocytes responsible for the disposal of all apoptotic cells and invading microorganisms. They become phagocytic when they encounter apoptotic cell corpses. Plasmatocytes are highly motile, use their actin-rich filopodia and lamellipodia to migrate from the head mesoderm to disperse across the embryo.

Question 31

How does JAK/STAT signalling occur in drosophila?

Answer 31

UPD directly binds DOME which brings HOP into proximity. JAK proteins phosphorylate the receptor creating binding sites for STAT proteins. STATs are phosphorylated, dimmerise and translocate to the nucleus to induce gene transcription.

Question 32

What are STATs like?

Answer 32

STAT proteins have an oligomerization domain, coiled-coil domain, DNA binding domain (contributes to nuclear localisation signal), linker domain, SH2 domain and trans-activation domain.
Activation of STAT family members requires transient association of the STATs with cytokine receptors.
Because of the size of STATs they have to be actively translocated into the nucleus.

Question 33

What are SOCS proteins?

Answer 33

Suppressors of cytokine signalling proteins. Induced by JAK/STAT pathway and inhibit STAT-mediated signal transduction (feedback inhibitors).
Includes SOCS1-7 and CISH.

Question 34

What is EPO and TPO?

Answer 34

EPO stands for Erythropoietin (also known as hematopoietin). A glycoprotein cytokine constantly secreted by the kidney which stimulates red blood cell production in the bone marrow for healthy RBC turnover. Receptor is EPO-R

TPO stands for Thrombopoietin is a glycoprotein hormone produced by the liver and the kidney to regulate the number of platelets. It stimulates the production and differentiation of megakaryocytes, the bone marrow cells that bud off large numbers of platelets. Receptor is cMPL

Question 35

Why might there be two bands when performing a western blot for m-cpl?

Answer 35

There are two different forms of m-CPL which will have two different molecular weights.

Question 36

Why is the mutation in CALR gain of function?

Answer 36

In the WT, the P domain inhibits the binding of c-MPL to the N domain. But in the mutant with the addition of the P interference domain which inhibits the P domain, negative regulation of c-MPL binding is prevented, allowing c-MPL to bind to the N domain.

Question 37

How does mutant CALR cause increased TPO cell proliferation?

Answer 37

Mutant CALR induced TPO-independent growth of TPO cells. This has found to be through preferential association with the TPO receptor, c-MPL. A change in the regulatory domains of mutant CALR, allows c-MPL to bind to the N domain of CALR. Mutant CALR then induces the phosphorylation of JAK2 and its downstream signalling, increasinig proliferation (can be blocked by JAK2 inhibitor) leading to myloproliferative neoplasms.