Leukaemia

Question 1

In to what categories can leukaemia be grouped?

Answer 1

Chronic or Acute, Lymphoid or Myeloid.

Question 2

What is the difference between chronic and acute leukaemia?

Answer 2

In Chronic leukaemias, the affected cells partly mature but fail to completely mature. The resulting cells are long lived and thus accumulate. Progression of this disease is slow so most patients are long lived.

Acute leukaemia is characerised by cells which cannot mature at all, so continue proliferating rapidly and so rapidly accumulate. Left untreated, those who develop this have a lifespan of months.

Question 3

What is the difference between lymphoid and myeloid leukaemia?

Answer 3

Lymphoid starts in the immature lymphocytes, whereas myeloid starts in early forms of myeloid cells.

Myeloid cells are cells that produce other blood cells (non-lymphocyte WBCs, RBCs, megakaryocytes).

Question 4

What is the difference between leukaemia and lymphoma?

Answer 4

Lymphomas are types of cancer that begin in lymphocytes but are not lymphoid leukaemias. They are characterised by tumour formation in lymph nodes and other organs, while leukaemias mainly affect the bone marrow and blood.

Question 5

How can the mutations seen in leukaemias be classed?

Answer 5

As mutations that are general to many cancers (eg p53, Ras) or ones specific to leukaemia/lymphoma.

Most leukamia specific genetic alterations are chromosomal translocations.

Question 6

How can chromosomal translocations lead to altered gene expression?

Answer 6

Juxtaposition of foreign genetic elements can have and effect due to the generation of chimeric fusion proteins or through an increase in expression due to the proximity of a powerful promoter/enhancer.

Upregulation due to proximity of a powerful Ig enhancer is common in Burkitt’s Lymphoma (Myc translocation).

Question 7

What is an example of a fusion oncoprotein?

Answer 7

BCR-ABL, a translocation specific to chronic myeloid leukaemia (CML).

In this, translocation of the abl gene from chromosome 9 to the region of chromosome 22 containing bcr produces the t(9;22) philadelphia chromosome.

Question 8

How does BCR-ABL expression lead to CML?

Answer 8

Expression of the BCR-ABL fusion protein in self-renewing haematopoietic stem cells (HSCs) which differentiate into common myeloid progenitors (CMPs) which then differentiate into granulocyte/macrophage progenitors (GMPs), which eventually form granulocytic cells.

CML is a biphasic disease, first characterised by the massive expansion of the granulocytic cells in the chronic phase due to BCR-ABL expression. Accumulation of additional mutation progresses it to the blast phase in which there is instead massive expansion of the earlier GMP cells.

Question 9

What is Abl? Describe its WT structure.

Answer 9

Abelson murine leukaemia viral oncogene homologue (Abl) kinase is a nuclear and cytoplasmic RTK involved in the regulation of differentiation, proliferation and adhesion, similar to Src.

It contains a N-terminal myristoylated cap responsible for membrane localisation, followed by SH3 and 2 domains. The N and C lobes that form the kinase domain follow, separated by a region containing Y412 which acts as a regulatory switch. There is then a long C-terminal single-exon-encoded region terminating in an actin binding domain.

Question 10

How is WT Abl kinase regulated?

Answer 10

It forms an intramolecular pseudotetramer with the SH3 and 2 domains binding the N and C kinase lobes. The myristoylated cap acts as a ‘latch’, coming from the SH3 domain and binding to the C-terminal C-lobe (the myristoyl group binding into a hydrophobic pocket). This inhibits the activity of the kinase.

Phosphorylation of the Y412 switch between the N and C lobes disrupts this structure, preventing the domains from interacting and inhibiting the kinase activity.

Question 11

What is WT BCR? Describe its domain structure.

Answer 11

The funtion of the wt gene is not yet clear.

It is known that it contains an N-terminal coiled-coil dimerisation domain, followed by a Ser/Thr Kinase domain, a DB-homology Rho G GEF domain and a GAP domain that targets Rac proteins.

Question 12

Describe the structure of the BCR-ABL fusion protein.

Answer 12

The N-terminal BCR region is spliced onto the C-terminal region of the Abl.

This removes the N-terminal myristolyated cap from Abl, removing its autoinhibition and making it consitituively active, while replacing it with a dimerisation domain that allows contant dimerisation of the RTK and hence continuous activity, also localising the protein to the cytoplasm to allow it to interacting with survival/proliferation pathways inc. Ras and PI3K.

Question 13

What are the two main variants of the BCR-ABL fusion protein?

Answer 13

p210 BCR-ABL contains the BCR DD and domains 1 and 2, fused with the Abl so that it contains all domains other than the N-terminal cap (including SH3). This is common in CML.

p190 BCR-ABL is the same, but lacking in the second BCR domain (the DB homology GEF domain). This is more common in acute lymphoblastic leukaemias (ALLs) and is a more active oncogene, thus leading to a more aggressive cancer.

Question 14

How is philadelphia chromosome-induced CML treated?

Answer 14

Imatinib is a small molecule ABL kinase inhibitor used in treatment, and is highly effective in the early phase when the fusion protein is constitutively active.

However, there is a high relapse rate in advanced/blast-crisis phase patients as the protein evolves mutations in the Abl kinase domain to prevent inhibitor binding. Several second generation inhibitors have been developed such as Nilotinib and Dasatinib.

Question 15

What is PML-RARα?

Answer 15

A fusion oncoprotein found in acute promyelotic leukaemia (APL - the M3 subtype of AML) caused by t(15;17).

Question 16

How does PML-RARα cause leukaemia?

Answer 16

This causes leukaemia by blocking myelopoiesis (differentiation into myeloid cells) leading to accumulation of leukaemic blasts.

Question 17

What is RARα?

Answer 17

The retinoic acid receptor alpha gene (RARα) - a nuclear receptor that when activated colocalises with RXR (retinoid X receptor) to signal to bind and repress genes with RARα response elements by recruitment of the NCoR/SMRT HDAC complex, repressing differentiation genes (and favouring proliferative ones).

Question 18

What is PML?

Answer 18

Promyelocytic leukaemia gene (PML) encodes a redox sensitive protein known to organise PML bodies (nuclear aggregates thought to be involved in transcriptional activation and p53 regulation - thought to be phase separated bodies).

Question 19

How is the PML-RARα fusion protein formed?

Answer 19

The RARα is a 462AA protein, with everything C-terminal for residue 61 being incuded in the fusion protein including the hinge and ligand binding region.

The N-terminal region of PML is spliced in, with the breakpoint in the 882AA protein being at either residue 395 or 552 producing two different PML-RARα fusion protein isoforms of either 796 or 954AA.

Question 20

How does the PML-RARα fusion protein act?

Answer 20

It binds to the RARα response elements more strongly than the RARα-RXR dimer, displacing it, and binding de novo sites. It has stronger repressive effects and is less sensitive to RA regulation, heightening the oncogenic effect of RARα.

It is also able to delocalise PML out of PML bodies, which is thought to have a role in self renewal and cell death regulation.

Question 21

How is PML-RARα APL treated?

Answer 21

Very high levels of Retinoic Acid cause PML-RARα to act as a transcriptional activator rather than repressor, and induces proteasome-mediated degradation of the fusion protein.

Arsenic trioxide (As2O3) is s highly toxic compound that leads to arsenic-induced apoptosis in the cells through activation of stress responses, mitochondrial toxicity and oxidative stress.

On its own As2O3 has a 70% cure rate, in combination with RA the cure rate is 90%. They are thought to work together to increase PML-RARα degradation further, making childhoos ALL the first cancer systematically cured by chemotherapy.

Question 22

How is PML-RARα degradation caused by combination therapy?

Answer 22

(a) As2O3 triggers PML/RARA degradation by the proteasome in PML nuclear bodies, via the SUMO mediated/ubiquitin-dependent pathway acting on the PML portion
(b) RA activates PML/RARA degradation by nuclear receptor negative feedback through proteasome interaction with the RARA portion.
(c) RA also induces cleavage by neutrophil elastase.
(d) Both RA and As2O3 target PML/RARA to autophagosomes

Question 23

What are three examples of high expression due to translocation of a gene to the locale of an enhancer in leukaemia?

Answer 23

This occurs naturally in the immunoglobulin loci, meaning that the heavy chain locus on chromosome 14 is often implicated.

In 100% of cases of Burkitt’s Lymphoma there is a t(8;14) translocation that puts Myc under the control of one such enhancer.

~80% of follicular lymphoma is caused by t(14;18) that increases expression of BCL2.

> 95% of Mantle cell lymphomas are the result of t(11;14) leading to increased CycD1 expression.

Question 24

What causes Burkitt’s Lymphoma?

Answer 24

This requires three seperate events to occur in order to cause the disease.

Infection with EBV leads to B-cell immortalisation and proliferation. Malarial T-cell immunosupression is required, and the t(8;14) translocation must also occur due to misfiring of the Ig gene rearranging machinery to lead to overexpression of Myc.

Hence this cancer has an epidemiology concurrent with the overlap in the distribution of EBV and malaria.

Question 25

Where is the Myc gene inserted in the Burkitt’s Lymphoma t(8;14) translocation?

Answer 25

The three-exon Myc gene is inserted within the J region segmentss of the heavy chain locus between 3 and 4. This allows the enhancer that follows the J-region to upregulate it.

Question 26

What effect does increased Myc signalling in Burkitt’s Lymphoma cause?

Answer 26

c-Myc promoted proliferation by stimulating transcription of genes that promote progression from G1 to S.

It’s TF activities also regulate glycolysis and the citric acid cycle, and both nucleotide and amino acid synthesis to promote cell growth.

Although the increased c-Myc activity does activate its p14-ARF/p53 mediated negative feedback mechanism it is insufficient to prevent hyperactivity, though they are prone to mutation to further increase myc activity.

Question 27

How does the follicular lymphoma translocation lead to cancer?

Answer 27

The t(14;18) translocation puts the apoptosis inhibitor Bcl-2 under the control of the powerful heavy chain locus enhancers.

Question 28

What are the components of the apoptosis regulatory network?

Answer 28

The apoptotic machinery is composed of both upstream regulators and downstream effector components (Adams and Cory, 2007).

The regulators, in turn, are divided into two major circuits, one receiving and processing extracellular death-inducing signals (the extrinsic apoptotic program, involving for example the Fas ligand/Fas receptor), and the other sensing and integrating a variety of signals of intracellular origin (the intrinsic program, more cancer relevant).

Each culminates in activation of a normally latent protease (caspases 8 and 9, respectively), which proceeds to initiate a cascade of proteolysis involving effector caspases responsible for the execution phase of apoptosis, in which the cell is progressively disassembled and then consumed,

Question 29

What are the components of the intrinsic apoptotic regulatory system?

Answer 29

The ‘‘apoptotic trigger’’ that conveys signals between the regulators and effectors is controlled by counterbalancing pro- and antiapoptotic members of the Bcl-2 family.

Bcl-2, along with its closest relatives (Bcl-xL, Bcl-w, Mcl-1, A1) are inhibitors of apoptosis, acting by supressing the OMM-embedded Bax and Bak.

Without this suppression they disrupt the membrane leading to the release of Cyt C and other pro-apop factors which activates Caspase 9 via Apaf-1 activation to begin the caspase cascade, whose myriad proteolytic actions begin the process of cell death.

Question 30

How does p53 stimulate apoptosis?

Answer 30

The anti-apop Bcl-2s are themselves inhibited by BH3-only proteins. p53 induces apoptosis by increasing ranscription of two BH3 proteins, noxa and puma.

Question 31

How does TNFa induce apoptosis?

Answer 31

TNFa receptors and other ‘death receptors’ activate FADD, which activates Caspase-8 to initiate the caspase cascade.

Question 32

How does the mantle cell lymphoma cause cancer?

Answer 32

The t(11;14) translocation puts CycD1 under the control of the powerful Ig enhancers. Overexpression of D1 leads to an increase in the number of CycD-CDK4/6 complexes which promote cell cycle entry by phosphoinhibition of Rb, allowing the cell to pass the restriction point.

Question 33

What are the main varieties of treatment option for leukaemia?

Answer 33

Chemotherapy - drug combinations including antifolates, corticosteroids and cytotoxins.

Stem cell transplantation - bone marrow transplant enables replenishment of marrow after irradiation destroys all cancerous marrow cells.

Immunotherapy - A recent experimental approach is to extract immunce cells and re-engineer them to target the cancer cells when they are re-introduced.