1. The fight against Cancer: can our immune system deliver a knockout blow?
Role of our immune system in Cancer prevention and development
A potential roll of an ‘autoimmune antigen’
in the treatment of Chronic Myeloid Leukaemia
Dr. David Pryce
Bangor University School of Medical Sciences

2. Our Immune System Innate immunity Adaptive immunity
No Memory
Memory
Protects the body from pathogens
Five major groups of potential pathogens:
Virus, bacteria, fungi, protozoa and helminths (worms).
208 viruses
538 bacteria
317 fungi
57 parasitic protozoa
287 worms
(CDC figures 2011)
‘The boy in the bubble’
David Phillip Vetter 1971 – 1984
Severe Combined Immuno-Deficiency (SCID)
Cause of death: Burkitt’s Lymphoma

3. Our Immune System How does the immune system recognise the bad guys?
Immune Cells and antibodies
‘survey’ our tissues and body

4. Our Immune System How does the immune system recognise the bad guys?
Histocompatibility complexes

5. Our Immune System Adaptive immunity The environment we live in
The set of adaptive immune cells each person produces is unique to them
Every immune cell set activates, develops and adapts to the ‘experiences’ our lives
The environment we live in
Who we choose as our ‘partner’
Who we ‘cohabitate’ with
’Positive outlook’ on life
The Aging process

6. Our Immune System Innate immunity Adaptive immunity Autoimmune Disease
Autoimmune disease can result if our immune inappropriately ‘attacks’ the body’s own cells
More than 80 Autoimmune diseases have been classified
Most are chronic and incurable
Most Autoimmune diseases effect Women more than men
Loss of regulation

7. Our Immune System and Cancer where’s the link?

8. This hypothesis lasted about
Cancer Research
The earliest recorded theory of Human cancer was based on the humoral theory of disease
Hippocrates (460–370 BCE).
He was postulated that the disease was due to an excess of “black bile”
‘Modern’ cancer research really began in the 19th century
Exposure to aromatic amines and bladder cancer
Snuff with nasal polyps
Soot with scrotal cancer
Reproductive factors with breast cancer
Set the stage for the identification of carcinogens chemical agents that can cause cancer
Rudolf Virchow: cancer is a disease of cells.
This hypothesis lasted about
1,900 years!

9. Cancer Research Our understanding in the 21st century
Rudolf Virchow was correct cancer is a disease of cells.
More precisely
Cancer arises due to Mutation(s) in….
Our Genetic code and/or
Our Epigenetic code
Mutation(s) can be Inherited and/or developed
Genetic Mutation(s)
Epigenetic Mutation(s)

10. Human Cancers
About 93% of our cells are ‘blood cells’: Red Blood Cells, platelets or bone marrow cells
At least 50 other types of nucleated cells make up the rest
(Bianconi et al., 2013)

11. Our Immune System and cancer – the link! Innate immunity Adaptive
‘Natural killers’
No Memory
B cells, T cells
Memory
Can our immune system ‘control’ Cancer?
Mutation(s)
Adapted from:

12. Cancer Treatments
Hanahan, D. and Weinberg, R. A. (2011) ‘Hallmarks of cancer: the next generation.’, Cell, 144(5), pp. 646–674. doi: /j.cell

13. Cancer Immunotherapy Immunotherapy…. ‘Conventional’ therapy….
‘Specific’ attack
Can stimulate Immune system
Can Help Target drugs
‘Conventional’ therapy….
Often Relatively non-specific
Radiation treatment
Chemotherapies

14. Monoclonal Antibodies
Well over 200 monoclonal antibodies have passed through some kind of clinical trial
AMA (generic) Naming convention
Monoclonal antibody list
Monoclonal antibodies: versatile platforms for cancer immunotherapy
Louis M. Weiner, Rishi Surana, Shangzi Wang
Nature Reviews Immunology 10, (May 2010) doi: /nri2744 Review

15. Research
Introduce some of the latest work from my research group
A potential roll of an ‘autoimmune antigen’ in the treatment of Chronic Myeloid Leukaemia

16. ‘Blood cancers’ Leukaemic Stem Cell? CML Mulitple myeloma
Myelogenous leukaemia
Acute myelogenous leukemia (AML)
Chronic myelogenous leukemia (CML)
Lymphocytic leukaemia
Acute lymphoblastic leukemia (ALL)
Chronic lymphocytic leukemia (CLL)
CML
Mulitple myeloma
plasma cells in bone marrow
form solid tumors.

17. Chronic Myeloid Leukaemia
Chronic Myeloid Leukemia (CML)
Develops from a mutated hematopoietic stem-cell/common myeloid progenitor
That contains a BCR-ABL ‘fusion gene’
8% of all leukaemias and 0.2% of all new cancer cases (UK 2013)
Incidence and prevalence gradually rising developed countries highest rates.
Chronic myeloid leukaemia (CML) is form of Non-Hodgkin lymphoma (NHL)19. World-wide CML incidence and prevalence are gradually rising, with developed countries observing the highest rates In 2013, CML cases accounted for 8% of all leukaemias and 0.2% of all new United Kingdom cancer cases23.
Current CML therapies. CML develops from an haematopoietic stem-cell with erythrocyte and granulocyte progenitor potential3,24 characterized by the presence of a key driver mutation; a balanced t(9;22)(q34;q11.2) chromosome translocation event Untreated, CML follows a tri-phasic disease pattern, with a median 5-year progression, starting from an initial chronic phase, in which most diagnosis are made, progressing through an accelerated phase to a final, usually fatal blast crisis phase. The discovery of tyrosine kinase inhibitors (TKIs) - that highly specifically inhibit the BCR-ABL kinase - has profoundly influenced the disease profile of BCR-ABL positive CML. Globally, the use of TKIs has reduced the annual CML mortality rate, from a pre-TKI rate of 10-20% to approximately 2%. Moreover, with full compliance to treatment regimes the estimated median 10-year survival can reach up to 80%28. As the mean age of CML diagnosis in the UK is 64.4 years for males and 66.5 years for females, this means successful TKI treatment can extend survivability to close to the normal expected lifespan. 
Requirement for ‘new’ therapies to enhance CML treatment. Despite the unprecedented and obvious clinical efficacy, several issues still remain with current TKI treatments. Prolonged therapy can result in disease relapse, due to occurrence of resistance mutationsreviewed in: 29. In addition, clinical trials indicate ‘successful’ therapy withdraw is possible only in a small percentage of patients, who have already undergone a prolonged TKI therapy regime, and interruption of treatment usually lead to disease relapse TKI CML treatment regimes are therefore usual ‘life–long’. NICE currently estimate the annual cost of first-line TKI (Imatinib) therapy at around £19,000 - £38,000, whilst second and third generation TKI treatments are 10-20% more expensive. NICE also recommend "Further good-quality studies are also needed to investigate the efficacy of Imatinib in combination with other treatment options”33 and there is a growing consensus that ‘new treatments for CML’ are urgently required1,34.

18. Chronic Myeloid Leukaemia
‘3–stage’ disease Initial chronic phase –to- blast crisis
Mean age of diagnosis: UK males, 66.5 females
Very successful drug therapy
Tyrosine kinase inhibitors (TKI)
Reduced the annual mortality rate 10-fold
Can extend survivability to close to the normal expected lifespan. 
Requirement for ‘new’ therapies to enhance CML treatment. Despite the unprecedented and obvious clinical efficacy, several issues still remain with current TKI treatments. Prolonged therapy can result in disease relapse, due to occurrence of resistance mutationsreviewed in: 29. In addition, clinical trials indicate ‘successful’ therapy withdraw is possible only in a small percentage of patients, who have already undergone a prolonged TKI therapy regime, and interruption of treatment usually lead to disease relapse TKI CML treatment regimes are therefore usual ‘life–long’. NICE currently estimate the annual cost of first-line TKI (Imatinib) therapy at around £19,000 - £38,000, whilst second and third generation TKI treatments are 10-20% more expensive. NICE also recommend "Further good-quality studies are also needed to investigate the efficacy of Imatinib in combination with other treatment options”33 and there is a growing consensus that ‘new treatments for CML’ are urgently required1,34.
Do we need more drugs?
Weisberg, E., Manley, P. W., Cowan-Jacob, S. W., Hochhaus, A., & Griffin, J. D. (2007). Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia. Nature Reviews Cancer, 7(5), 345–356.

19. Chronic Myeloid Leukaemia
TKI treatment usual ‘life–long’.
Compliance to treatment regimes essential
Treatment withdrawal requires minimum 2-year of therapy
≅20% patients serious side effects/quality of life issues
Resistance: BCR-ABL mutations, and ‘other’ pathways
annual cost of first-line TKI (Imatinib) therapy £19,000 - £38,000
2nd and 3rd generation TKIs at least 10-20% more expensive
"Further good-quality studies are needed”
Requirement for ‘new’ therapies to enhance CML treatment.
Weisberg, E., Manley, P. W., Cowan-Jacob, S. W., Hochhaus, A., & Griffin, J. D. (2007). Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia. Nature Reviews Cancer, 7(5), 345–356.

20. Chronic Myeloid Leukaemia
Treatment summary
TKI therapies are remarkable success story, but not
perfect drugs
N.I.C.E recommendations
"Further good-quality studies are needed to investigate
the efficacy of Imatinib in combination with other
Treatment options”
N.I.C.E. are not alone!
There is a growing consensus that new treatments for
CML are urgently required 1,2
1 Holyoake, T. L. & Helgason, G. V. Do we need more drugs for chronic myeloid leukemia? Immunological Reviews 263, 106–123 (2015).
2 Leukemia, E. I. C. M. et al. The price of drugs for chronic myeloid leukemia (CML) is a reflection of the unsustainable prices of cancer drugs: from the perspective of a large group of CML experts. Blood 121, 4439–4442 (2013).

21. Our Immune System Innate immunity Adaptive immunity
Autoimmunity in Cancer therapy
Reactivating an ‘exhausted immune may reinvigorate the immune ‘attack’ on specific targets
Amazing successful results using monoclonal antibodies therapies that target key ‘immune checkpoints’
Remove the brakes!

22. The TROVE2 protein YRNAs 4 know genes, numerous psuedogenes Y-RNA
N-term ‘surface’
ssRNA
Central cavity
Conserved region - required for DNA replication
Up-regulated in tumours
‘small- YRNAs’ biomarkers
Alu element RNAs (human), 5S rRNA (Xenopus), U2 snRNAs (mouse - ES cells)
Roles with Ro60
RNA quality control
Molecular associations with other protein
Ro60 Surfaces that Interact with RNA Molecular surface representation of Ro structure (Xenopus laevis)*
Y-RNA interaction regions are coloured green
single-stranded RNA interaction regions are coloured teal. *(78% identical to human Ro60)
Adapted from
Stein et al (2005). Structural insights into RNA quality control: The Ro autoantigen binds misfolded RNAs via its central cavity. Cell 121, 529–539.

23. TROVE2 Autoimmune Epitopes and Disease
Yellow
correspond to human Ro60 amino acids.
Ro60 ‘autoantigen’
Components:
TROVE2 (Ro60, SSA2)
TRIM21 (Ro52, SSA1)
YRNAs
Autoantibodies against TROVE2
Detected in –
Sjögren’s syndrome (38–90%)*
Systemic Lupus Erythematosus (24–60%)*
Subacute cutaneous lupus erythematosus (70–100%)*
The ‘first’ autoantibodies detected in these diseases - up to 4 years prior to patent disease
Detected in number of other ‘systemic’ autoimmune disease
*Depends on study and techniques used for detection
Sjögren’s Major epitope
Epitopes recognized by anti-Ro60 autoantibodies overlap with RNA binding regions
Note – additional surfaces that contact the bound Y-RNA, or that contact misfolded RNAs, may not yet have been identified.
Adapted from
Wolin and Reinisch (2006). The Ro 60 kDa autoantigen comes into focus: Interpreting epitope mapping experiments on the basis of structure. Autoimmunity Reviews 5, 367–372.
Lupus
Major Epitope Major Epitope2 -
Depending on the study and the techniques used for detection, anti-Ro60 antibodies have been found in 38–90% of patients with Sjogren’s syndrome, 24–60% of patients with systemic lupus erythematosus and 70–100% of patients with subacute cutaneous lupus erythematosus
Monneaux and Muller, S. (2002). Epitope spreading in systemic lupus erythematosus: Identification of triggering peptide sequences. Arthritis Rheum. 46, 1430–1438.

24. TROVE2 Anti-Sense RNA depletion
Main Objectives
siRNA(s) with most effective TROVE2 knockdown
Optimize their transfection per cell line
Validate specificity of anti TROVE2 primary antibodies
Determine ‘viability’ of knockdown phenotype
siRNAs
Anti-sense oligonucleotides (ASO)
microRNAs (miRNA)

25. TROVE2 Anti-Sense RNA (CML cell lines)
‘knocking down’ TROVE2
in CML Blast Crisis cell lines results in increased susceptibility to TKI drugs

26. Thank you for listening
The fight against Cancer: can our immune system deliver a knockout blow?
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