A Glimpse into the Genetics of CLL

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Presentation transcript:

A Glimpse into the Genetics of CLL By Lara Makewita Ph.D. Student

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

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

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.

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

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

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

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

Balance is Key Cell Production Cell Death Normal Cells

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

Balance is Key Cell Production Cell Death Leukaemic Cells

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

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

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

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

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

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

without D gene segments V 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

V D J C V D J C

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

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

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

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.

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% 99.99 - 98% <98% IGHV unmutated CLL IGHV mutated CLL Percentage Similarity to Germline IGHV

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…

Chromosomal Abnormalities 1 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

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!

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

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

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

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

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

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

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

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.

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%

A Little About My Research: Epigenetics in CLL

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???

Gene Expression Central dogma of biology

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

Imagine… LIFE

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

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

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

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

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

Thank you for listening!