1. Co-supervisor: Prof Richard Lock
The BET Bromodomain inhibitor ( JQ1) and Histone Deacetylase inhibitor (Panobinostat) induce synergistic anticancer effects by repressing LIN28B and N-Myc expression
Dr Jeyran Shahbazi
Supervisor: Dr Tao Liu
Co-supervisor: Prof Richard Lock

2. Neuroblastoma Average age of diagnosis: 18 months
Most common childhood solid tumour
In the nervous system outside of the brain
Average age of diagnosis: 18 months
Accounts for 15-20% of childhood cancer deaths
Patients are classified into 5 stages based on
Patient age at time of diagnosis
Tumour biology
MYCN amplification status
Neuroblastoma is the most common solid tumor in children under the age of five, originating from neural crest cells in the peripheral nervous system.
The average age of diagnosis is 18 months and is responsible for 15% of childhood cancer deaths.
Neuroblastoma has diverse clinical outcomes, ranging from spontaneously regression with little to no treatment or highly metastasis despite intensive treatment.
International Neuroblastoma staging sysmtem, developed in mid 90s, classified patients into 5 stages based on age at time of diagnosis, tumor biology and MYCN amplification.
Stage 1 and 2 have localised tumours which can be removed with surgery. Stage 3 would require surgery as well as chemotherapy. However stage 4 presented with tumour metastasis to bone, liver, lymph node and other organs and is considered high risk with frequent replapse evern after intenstive chemotherapy.
This has been attributed to molecular differences among tumors with key factor being tumor amplification of the N-myc gene.
Around 25% of neuroblastoma cases being caused by N-myc overexpression.
A Kaplan–Meier survival curve of infants less than 1 year of age with metastatic neuroblastoma show EFS of only 10%.

3. Stage 1 or 2 : Low Risk

4. Stage 3 or 4: High Risk
…Strongly associated with high MYCN amplification

5. What is MYCN? Mammalian oncogene Transcription factor
Expressed in wide variety of cancers
Over 200 copies of MYC found in neuroblastoma
25% of neuroblastoma is caused by MYCN oncogene amplification
N-myc is a member of the MYC Oncogene family which includes c-Myc. Expressed during embryogenesis and specifically during early cell differentiation stages in the parts of the central nervous system, kidney and hair follicles.
All myc family proteins contains a transactivation domain, basic helix-loop-helix motif, and Leucin-zipper, allowing N-MYC to heterodimerize with MAX, a small ubiquitously expressed transcription factor.
This N-myc/Max dimer influences a multitude of cellular processes through regulation of gene transciption. The NMYC-Max dimer binds to the E-Box sequence of the target gene promoter and recruits histone acetyl transferases such as Tip60- and Cdk9 complexes leading to an open chromatin state. The MYC/Max dimer then promotes phosphorylation of the C-terminal domain of RNA polymerase II by Cyclin dependant kinase 9, resulting in transcription of the target gene.

6. ~ ~ What does MYC do? Cancer cells Normal cells MYC MYC Active gene
Promoter
Active gene
Promoter
MYC ~ ~
Transcriptional activation of the genes
Well MYC is a transcription factor that acts as a master regulatory protein in cancer
Basically, It s a protein synthesised by cells that turns genes on, a gene activating protein! A light switch
MYC doesnot function by itself, it partners up with another protein MAX
When they bind, they intertwind themselves as hetrodimer and sit down on DNA throughout the nucleolus
Where ever they sit, they employ the other elements of transcription complex and initiate the transcription of the genes.
Evolution has positioned MYC binding site throughout the nucleolus adjacent to the genes that control growth.
It is physiologically helpful to have myc to drive the formation of new blood cells
Cancer steals MYC, inappropriately activates and maintains it in ON position and builds up on genes such that it causes the cells to forget to stop growing.
Today therapeutic strategies to target Myc does directly not exist, only the perception regarding its durability,
Structural studies of C-myc failed to identify a hydrophobic pocket into which an organice chemist might fashion a small molecule
Cell growth and amplification

7. Therapeutic approach targeting MYC
Today, there is No therapeutic strategies to target MYC gene
Structural analysis of N-Myc protein has failed to identify a hydrophobic pocket into which a small inhibitory molecule could be designed.
MYC protein structure

8. Alternative approach: BET inhibitors
MYC
BRD4
Promoter
Active gene ON
BET inhibitor
MYC X
BRD4
Recently, quite a few studies have shown that Bromodomain proteins, plays a major role in modulating MYC transcription.
The poposed model is that BRDs particlarly BRD4 recognise acetyl lysine reside on the histone and therefore recruits the other elements of transcription to the promoter of MYC; therefore activating the growth genes.
Promoter
Active gene
OFF

9. BET Inhibitors Small molecules Down-regulates MYC
Down-regulates MYC target genes
Shown promising effect against wide range of cancer
NUT midline carcinoma
Medulloblastoma
Neuroblastoma
Burkitt`s lymphoma
Myeloma
IBET762 Currently in clinical development for treatment of Leukaemia and lymphoma

10. BET inhibitor (+)-JQ1 reduces neuroblastoma cell proliferation

11. BET inhibitor (+)-JQ1 down-regulates N-Myc transcription
SK-N-BE(2) cell line
Kelly cell line
(+)-JQ1 (µM)
(+)-JQ1 (µM)
N-Myc
Actin 1
0.8
0.6
0.4
0.2 1
0.8
0.6
0.4
0.2
N-Myc mRNA expression
fold change
N-Myc mRNA expression
fold change
(+)-JQ1 (µM)
(+)-JQ1 (µM)

12. Combination therapy
No research has yet shown complete cancer remission with the use of BET Inhibitors alone
Histone Modification group has identified novel interplays among HDAC1, HDAC2, HDAC5, SIRT1 and Myc oncoproteins
Combination therapy of BET Bromodomain inhibitors with HDAC inhibitor
Panobinostat
Highly potent non-selective HDAC inhibitor
minimum toxicity on normal cell
Recently passed FDA approval to be used in clinics

13. Combination treatments synergistically reduce neuroblastoma cell proliferation

14. lysine hyperacetylation induced by treatment with Panobinostat increases the dependency of Neuroblastoma cells on BET protein regulated transcription of the oncogenes
150
100 50
Cell Viability %
LBH JQ

15. Combination treatment synergistically reduce neuroblastoma cell proliferation

16. Combination Therapy Enhances the effect on the Expression of Target Genes
Control
JQ1
Panobinostat
Combination
Up-regulated
JQ1
Panobinostat
Combination
FC > 2
283 8 80
197 40 25 1
JQ1
Panobinostat
Combination
Down- regulated
FC > 2
660 5 48
314 68
110 1
Just mention that a synergistic dose was selected for further mechanistic analysis.

17. Genes most significantly modulated by combination therapy
Gene name
Fold Change
Up or Down
Comment
TFAP2B
10.5
Down
Transcriptional activator and repressor
MIR7-3HG
7.75 up
miRNA
ST8SIA3
7.07
WNT ligand biogenesis
NPY2R
6.7
affects tumor vasculature
LIN28B
5.2
Supressor of miRNA
NSDP
6.5
Activates ERK-Mediates proliferation pathway in Neuroblastoma
ATP1B2
5.62
Mediates cell adhesion of neurons and astrocytes
Bcl-2
6.4
supresses apoptosis
HEXIM1
3.43
Cause growth inhibition and promotes neuronal differentiation
RUNX1T1
2.61
involved with MYCN gene development

18. LIN28B RNA binding protein Increases MYNC protein expression
Induce neuroblastoma in neural crest
High LIN28B expression is associated with poor patient prognosis
LIN28B expression is positively correlates with MYC expression

19. Combination therapy synergistically reduced Bcl-2, N-Myc and LIN28B protein expression
SK-N-BE(2)
Kelly
Ctrl JQ LBH Combo
Ctrl JQ LBH Combo
Bcl-2
N-Myc
LIN28B big isoform
LIN28B small isoform
Actin

20. Combination treatment induce apoptosis in neuroblastoma cells
Panobinostat (10nM)
JQ1 ( 1µM)
Control solvent
Combination
JQ1 ( 1µM) + Panobinostat (10nM) 4%
10%
18%
56%
Kelly
7AAD stain 1% 8%
18%
65%
SK-N-BE(2)
Make sure people understand these histograms! Explain them!
Show kelly and comparison!
Annexin VI stain

21. JQ1 ( 2µM) + Panobinostat (20nM)
Combination treatment does NOT induce apoptosis in normal fibroblast cells
Panobinostat (20nM)
JQ1 ( 2µM)
Control solvent
Combination
JQ1 ( 2µM) + Panobinostat (20nM)
3.2%
3.6%
8.7%
8.4%
7AAD stain
Annexin VI stain

22. Combination therapy significantly reduced tumour progression in vivo
Spend time on this!

23. Combination therapy significantly reduced MYC protein expression in mice tumour tissues
Control solvent
JQ1 (50mg/kg)
Panobinostat (7.5mg/kg)
JQ1 + Panobinostat

24. BET bromodomain proteins, BRD3 and BRD4 up-regulate LIN28B mRNA and protein expression
LIN28B long isoform
Actin
LIN28B short isoform
BRD3
BRD3 SCR BRD3 siRNA siRNA-2
SK-N-BE(2)
Kelly
BRD4
BRD4 SCR BRD4 siRNA siRNA-2
BRD3 SCR BRD3 siRNA siRNA-2
BRD4 SCR BRD4 siRNA siRNA-2

25. Bromodomain proteins bind directly to LIN28B promoter and enhancing its activity.
ChIP assay
Luciferase assay

26. Summary
Combination therapy significantly prolonged neuroblastoma tumour progression
Combination therapy significantly reduced N-Myc protein expression in neuroblastoma bearing mice
Bromodomain proteins, BRD3 and BRD4 bind directly to LIN28B promoter region, up-regulating its expression

27. With Thanks to…. My Supervisors: Histone Modification Group
Dr Tao Liu
Prof Richard Lock
Histone Modification Group
Children`s Cancer Institute
School of Biotechnology and Bio-molecular Sciences

28. Abstract
Patients with neuroblastoma associated with MYCN oncogene amplification experience a very poor prognosis. BET bromodomain inhibitors are emerging as one of the most promising novel classes of anticancer agents by blocking the BET bromodomain proteins BRD3 and BRD4 from activating transcription of oncogenes such as MYC and MYCN. However, treatment with BET bromodomain inhibitors alone does not result in cancer remission. Here we show that BRD3 and BRD4 directly bound to the LIN28B gene promoter and activated LIN28B gene transcription, and that knocking down LIN28B expression reduced the expression of N-Myc protein, but not N-Myc mRNA. Combination therapy with the BET bromodomain inhibitor JQ1 and the histone deacetylase inhibitor panobinostat synergistically suppressed LIN28B gene expression, reduced N-Myc mRNA expression to the same extent as JQ1 treatment alone, but considerably and synergistically reduced N-Myc protein expression. JQ1 and panobinostat induced synergistic growth inhibition and apoptosis in neuroblastoma cells, but not normal non-malignant cells in vitro. Importantly, in neuroblastoma-bearing mice, JQ1 and panobinostat combination therapy synergistically and considerably reduced N-Myc protein expression in tumor tissues and blocked tumor progression. Our findings have therefore identified a potential strategy to reduce N-Myc onco-protein expression and a novel therapeutic approach for the treatment of aggressive neuroblastoma -  

29. Results to show
BRD3 and BRD4 directly bound to the LIN28B gene promoter and activated LIN28B gene transcription, and that knocking down LIN28B expression reduced the expression of N-Myc protein, but not N-Myc mRNA
Combination therapy with the BET bromodomain inhibitor JQ1 and the histone deacetylase inhibitor panobinostat synergistically suppressed LIN28B gene expression, reduced N-Myc mRNA expression to the same extent as JQ1 treatment alone, but considerably and synergistically reduced N-Myc protein expression
JQ1 and panobinostat induced synergistic growth inhibition and apoptosis in neuroblastoma cells, but not normal non-malignant cells in vitro
Importantly, in neuroblastoma-bearing mice, JQ1 and panobinostat combination therapy synergistically and considerably reduced N-Myc protein expression in tumor tissues and blocked tumor progression.

30. The RNA binding protein: LIN28B
Regulate the expression of let-7 microRNA family during embryogenesis
LIN28B down-regulates let-7 pre-microRNAs, which increases MYCN protein expression
Targeted overexpression of LIN28B to the developing neural crest of mice induces neuroblastoma
Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers.
Lin28A recruits a TUTase (Zcchc11/TUT4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a Zcchc11-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts are restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors, whereas Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function and have implications for the development of new strategies for cancer therapy.

31. Role of BRDs in MYC transcription
Ac-Lys
Growth ON
Ac-Lys
MYC
Major function of BET bromodomain BRD4 is the recognition of acetylated histones found in the transcriptionally active regions of chromatin, which promotes the recruitment of transcriptional activators.
BRD4-NUT fusion protein is actually oncogenic.
Recently, quite a few studies have shown that Bromodomain proteins, plays a major role in modulating MYC transcription.
The poposed model is that BRDs particlarly BRD4 recognise acetyl lysine reside on the histone and therefore recruits the other elements of transcription to the promoter of MYC; therefore activating the growth genes.
BRDs
RNA polymerase
Transcription factor

32. Bromodomain inhibitors
Ac-Lys
Growth OFF
Ac-Lys
MYC
Bromodomain and extra-terminal (BET) proteins recognise specific acetylated lysine residues in the N-terminal tail of histones
Initiate transcription by recruiting transcriptional regulators
MYC target gene transactivation is associated with increased histone lysine acetylation
Members of the BET family, including BRD2, BRD3, BRD4, and BRDT, modulate gene expression by recruiting transcriptional regulators to specific genomic locations
The bromodomain and extra terminal (BET) family of proteins BRD3 and BRD4 have recently been shown to play critical roles in c-Myc gene transcription,
BRD3/BRD4 inhibitors JQ1 and I-BET151 considerably reduce c-Myc gene and
N-Myc gene transcription.
BRD4
RNA polymerase
Transcription factor
BET inhibitor