1. A Glimpse into the Genetics of CLLBy
Lara Makewita
Ph.D. Student

2. About Me and Our Group! Ph.D. student from the University of
Southampton at the Faculty of Medicine
A large CLL research group of which there
are 7 principal investigators leading research
My supervisor  Professor Jonathan Strefford
Our research group  focus on CLL (epi)genomics
Where we have a large CLL research group led by 7 Pis focusing on different research branches of CLL biology

3. Introduction
Leukaemia – a word coined in German from the Greek words leukos (white) and haima (blood).
Defined as a cancer of the blood which originates in the bone marrow
Loss of control of normal regulation mechanisms such that too many immature or abnormal white blood cells are made
Can normally identify it due to the loss of control of normal cell regulation which can lead to uncontrolled proliferation of cells

4. The Cell
Basic building blocks of all living things  over 37 trillion cells in your body!
Nucleus hosts our hereditary material i.e. DNA
I am only highlighting the nucleus here however there are several other important components in the cell that allow it to function properly, for example the mitochondria and the cytoplasm.

5. In the Nucleus
In the nucleus we have chromosomes. These molecules are made from DNA that has been tightly wound around proteins to form chromatin. This chromatin is then tightly wound further to make a chromosome. We have 23 pairs of these chromosomes in our body

6. In the Nucleus
And they look like this in real life!

7. Cell Cycle Cells follow the circle of life too!
Extremely important that this cycle is regulated properly for the well being of individuals
Mitosis
Phase 1
Cell Growth
So just like every living organism, cells must follow the circle of life too! Every cell must go through this cycle to produce more cells and this is how cell proliferation occurs. It is important that the cell follows this cycle properly as incorrect regulation can lead to the event of abnormal cells
Phase 3
Cell Growth Ch
Phase 2
Make more DNA!!! Ch

8. Balance is Key Cell Production Cell Death Normal Cells
So like all things in life, things must be kept in balance. So here we have a scale that compares the event of cell production against the event of cell death. In normal cells these events would be in balance and therefore the scale doesn’t move
Normal Cells

9. Balance is Key
Cell Production
Cell Death
Normal Cells

10. Balance is Key Cell Production Cell Death Leukaemic Cells
However in leukaemia cells, or any cancerous cell for that matter, these two components are not always in balance. We can observe a high number of proliferative events whereas there will be a normal rate of cell death.
Leukaemic Cells

11. Balance is Key
Cell Production
Cell Death
Leukaemic Cells

12. Balance is Key Cell Production Cell Death Leukaemic Cells
However, it is also possible that the cell death component is deficient. So therefore we have normal cell production rate but less cell death taking place, and therefore the scales still tip towards cell production
Leukaemic Cells

13. Balance is Key Cell Production Cell Death Leukaemic Cells
However, it is also possible that the cell death component is deficient. So therefore we have normal cell production rate but less cell death taking place, and therefore the scales still tip towards cell production
Leukaemic Cells

14. Chronic Lymphocytic Leukaemia (CLL)
The most common form of leukaemia in adults in the western world
It arises from B lymphocytes  a type of white blood cell
B lymphocytes are an important part of the immune system  gives the immune system memory to fight against known enemies in the bodies!!
Has a B-cell receptor (BCR) which allows them to recognise molecules or communicate with other cells

15. B Lymphocyte B-cell receptor B cell Molecule of interest Nucleus
BCR key to B cell biology.

16. B-cell Development BONE MARROW GERMINAL CENTRE Y Y Y Y Y Y Y
Plasma B cell
Naïve CD5+ B cell Y
Legend
Memory B cell Y
B-cell receptor (BCR)
Antigen
B-cell development

17. IG Genes IG  Immunoglobulin genes
A group of genes that help the BCR differentiate to adapt to a particular target
Makes the BCR more specific to a particular stimulus by undergoing several small programmed mutations

18. without D gene segmentsV V V V V D D D D D J J J J J C
IG Heavy Chain (IGHV):
with D gene segments V D J C V V V V V J J J J J C
How the IGHV genes are mutated
IG Light Chain
(IGLV):
without D gene segments V J C

19. V D J C V D J C

20. These BCRs are found on the cell membrane of B cells V D J C V D J C
These BCRs are found on the cell membrane of B cells
Naïve B cells that have not bound to foreign molecules will not undergo this process C

21. IGHV Genes IGHV genes code for part of the BCR
Very important for the proper formation of the BCR
BUT
CLL cells can arise before or after this adapting process can take place

22. Extremely different clinical outcomes
B-Cell Development
BONE MARROW
GERMINAL CENTRE Y Y Y Y Y
Plasma B cell
Naïve CD5+ B cell Y
Legend
Memory B cell Y
B-cell receptor (BCR)
Antigen
B-cell development
CLL transformation Y Y
Extremely different clinical outcomes
But why?
IGHV unmutated CLL
IGHV mutated CLL

23. IGHV Genes
Studies have established a clinico-biological feature of CLL by assessing the mutational status of the IGHV genes
Mutated IGHV CLL cases, M-CLL (≤98% similarity to the germline IGHV gene)
M-CLL is likely to have derived from memory B cells
Unmutated IGHV CLL cases, U-CLL (≥98% similarity to the germline IGHV gene)
U-CLL is likely to have stemmed from naïve B cells
Different studies have helped to establish differences due to the mutational status of IGHV genes and also identify the origins of these CLL cells. CLL cells that have undergone all or some of this process will be classed as mutated CLL or m-CLL and likely derived from these memory B cells. CLL cells that have not undergone this process will be classed as unmutated CLL or u-CLL and likely derived from naïve B cells.

24. Percentage Similarity to Germline IGHV
B-Cell Development
BONE MARROW
GERMINAL CENTRE Y Y Y Y Y
Plasma B cell
Naïve CD5+ B cell Y
Legend
Germline IGHV refers to the genes before alteration. The more similar the CLL cell is to the germline IGHV genes, the more likely it has not undergone any BCR specialisation
Memory B cell Y
B-cell receptor (BCR)
Antigen
B-cell development
CLL transformation Y Y
100% %
<98%
IGHV unmutated CLL
IGHV mutated CLL
Percentage Similarity to Germline IGHV

25. However…nothing is as simple as this in biology…
IGHV Genes
U-CLL cases are more likely to follow a more aggressive disease course
M-CLL cases are more likely to follow a more indolent disease course
However…nothing is as simple as this in biology…

26. Chromosomal Abnormalities1 2 3 4 5 6 7
Deletion 1 2 3 4 5 6 7
Insertion 1 2 3 4 5 6 7
Translocation 1 2 3 4 5 6 7
Duplication 1 2 3 4 5 6 7
Pericentric
Inversion 1 2 3 4 5 6 7
Paracentric
Main one in CLL is deletion events in chromosomes

27. Checkpoint Proteins in the Cell Cycle
Mitosis
Phase 1
Cell Growth
Essential in the cell cycle  prevents the cell cycle from continuing past checkpoints if something is wrong
Important that proteins facilitate these checkpoints
Activates DNA damage repair proteins
Or initiates cell death
Phase 3
Cell Growth Ch
Phase 2
Make more DNA!!! Ch
It is essential that the checkpoints in the cell cycle are enforced!

28. Checkpoint Proteins TP53 TP53  gene that codes for the p53 protein
Located on chromosome 17
Mitosis
Phase 1
Cell Growth
Phase 3
Cell Growth 17 Ch
Phase 2
Make more DNA!!! Ch

29. Checkpoint Proteins TP53
Mutations in this region can cause the production of a defunct protein
Mitosis
Phase 1
Cell Growth
Phase 3
Cell Growth 17 Ch
Phase 2
Make more DNA!!! Ch

30. Checkpoint Proteins TP53
Loss in this region will cause no protein production
Mitosis
Phase 1
Cell Growth
Phase 3
Cell Growth 17 Ch
Phase 2
Make more DNA!!! Ch

31. Checkpoint Proteins ATM ATM  gene that codes for the ATM protein
Located on chromosome 11
Mitosis
Phase 1
Cell Growth
Phase 3
Cell Growth 11 Ch
Phase 2
Make more DNA!!! Ch

32. Checkpoint Proteins ATM
Mutations in this region can cause the production of a defunct protein
Mitosis
Phase 1
Cell Growth
Phase 3
Cell Growth 11 Ch
Phase 2
Make more DNA!!! Ch

33. Checkpoint Proteins ATM
Loss in this region will cause no protein production
Mitosis
Phase 1
Cell Growth
Phase 3
Cell Growth 11 Ch
Phase 2
Make more DNA!!! Ch

34. Checkpoint Proteins Faulty proteins are not functional
Checkpoint in cell cycle cannot be enforced
Abnormal cells will be continually produced
TP53 mutations more often seen in del17p CLL patients
ATM mutations more often seen in del11q CLL patients
Mitosis
Phase 1
Cell Growth
Phase 3
Cell Growth Ch
Phase 2
Make more DNA!!! Ch

35. FISH – A Cytogenetic Technique
FISH - Fluorescence In-Situ Hybridisation
Technique used to identify the presence of specific DNA sequences
This technique labels DNA with fluorescent dye for a particular sequence/segment. IF the sequence/segment is there, it should glow. IF it’s not, then there will be now glowing…how del17p is identified.

36. FISH – A Cytogenetic Technique
Can identify if and which chromosomes have changed e.g. can observe deletions, inversions etc.
Clinicians use this technique to identify these changes and categorise patients to decide the appropriate treatment going forward
Genetic Abnormality
Prognosis
Associated with M-CLL or U-CLL
Prevalence
Deleted 13q arm (Del13q)
Good
M-CLL
~50%
Deleted 11q arm (Del11q)
Poor
U-CLL
~20%
Deleted 17p arm (Del17p)
~10%
Trisomy 12 (+12)
Intermediate
~16%

37. A Little About My Research: Epigenetics in CLL

38. What is she talking about???
What We know Already
CLL is a B cell cancer
Varied outcomes between patients
Due to various abnormalities in the genes e.g. U-CLL and M-CLL
Due to external factors to DNA that cause changes in gene expression
Looking at the epigenomics of CLL
What is she talking about???

39. Gene Expression
Central dogma of biology

40. Epigenetics
The study of changes in organisms caused by modifying gene expression rather than the alteration of the DNA sequence itself
External factors that cause the change in the shape of DNA to allow or prevent gene expression.
Gene Expression
No Gene Expression

41. Imagine…
LIFE

42. Actors/Actresses Script Epigenetic Director
Imagine the human life to be a film; then, the cells in our bodies would be actors and actresses following a script called “DNA”. The script will have certain parts that are key to forwarding the plot – we can liken these parts to genes. Epigenetics, then, would be the director – they can change and remove certain scenes and dialogue without changing the wording of the script. So ultimately, the director releases the final cut of the film. Epigenetics is essential to our lives
Epigenetic Director

43. Epigenetic Mechanism: Chromatin Modelling
Gene Expression
No Gene Expression Activity Possible
One mechanism is how this chromatin molecule moves and changes shape due to external factors to allow gene expression

44. My Work CLL has several sub-types
Want to improve our understanding of the interactions the surrounding environment of the cell has on the DNA of CLL cells
Identify active genes in stimulated CLL cells using research methods
Potential targets for future therapeutics
Have used methods that can identify these open regions in chromatin in the absence/presence of external stimuli
BCR pathway, BTK, ibtrubnib

45. My Work Analyse these opening regions using computational tools
Can take some of these regions and validate in the lab for future work in the clinic
Epigenetics will provide some insight to other ways gene regulation occurs in CLL
Will provide other prognostic markers by the means of identifying interacting molecules in the cell environment
Ever-evolving technology and progression in this field, so beneficial for many diseases in the long-run

46. Acknowledgements University of Southampton Bournemouth Hospital
Professor Jonathan Strefford
Dr Dean Bryant
Dr Jane Gibson
IRIDIS Team - IRIDIS High Performance Computing Facility
Bournemouth Hospital
Professor David Oscier
Dr Renata Walewska
Dr Helen McCarthy
Bournemouth Leukaemia Fund

47. Thank you for listening!