EZH2 Inhibition in Lymphoma B-Cell Lines

CFCE 6/27/14Aditi Rao

Background• What is epigenetics?

– Epigenetics means above the genome; it is essentially the turning on and off of genes

• It is the alteration of DNA, protein, or RNA, that changes how these molecules function and/or their regulation, without there being any effect on the primary sequences of these molecules

• Epigenome determines what cells should be: epigenetic control is what partly regulates the fate of cells since they all have the same DNA

• Epigenetic modifications examples– Chemical modifications of histones that directly affect gene

expression (methylation and acetylation are most common) Histones: grab the DNA and tighten or loosen to control gene expression known as epigenetic control

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Background:EZH2 mutations and B-Cell

Lymphoma 1• EZH2 is an enzyme encoded by the EZH2 gene• Its prominent role is mainly as a gene silencer it adds

three methyl groups to lysine 27 of histone 3 (H3K27)• Over expression/over activity of this gene cause more of the

functional EZH2 methylated enzyme, and this can cause tumors because of the over abundance of H3K27

• As an example, histone methylation can silence the expression of tumor suppressor genes (represses gene expression) and causes cancer

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Background: EZH2 mutations and B-Cell Lymphoma 2

• Two mutations in the enzyme are Y641 and A677, this means that on the 641st amino acid in the enzyme there is a mutation in the codon for tyrosine and on the 677th amino acid in the enzyme there is a mutation in the codon for alanine– These mutations cause an increase in EZH2 activity

• These are somatic heterozygous mutations that can cause diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma

• These mutations change the enzyme making it bind to other substrates which causes an imbalance in the H3K27 methylation

• The Y641 mutation is most frequent 4

Background:EZH2 mutations and GSK 126

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• It is hypothesized that inhibition of EZH2 methyltransferase will be an effective treatment for the lymphomas bearing EZH2 mutation

• So in a paper published in 2012*, a drug – GSK 126 was used to see if EZH2 inhibition is effective in treating models of lymphoma caused by EZH2-activating mutations

• GSK 126 is a potent, selective small molecule inhibitor of wild type and mutant ezh2 methyltransferase

• It was found that GSK126 inhibits excessive growth of cell lines and diffuse large B-cell lymphoma (DLBCL) xenografts bearing EZH2 mutation in mice

*“EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations.” http://www.ncbi.nlm.nih.gov/pubmed/23051747

Experimental Background

• To see the effect of GSK 126 on two types of cancer cell lines: RL (which is not sensitive to the drug) and Pfeiffer which is sensitive to the drug according to the published paper

• RL and Pfeiffer were used because they are B-Cell lymphoma cell lines and both have mutations in EZH2 that cause them to be hyper active

• We wanted to confirm the results published• According to the paper the IC50 of RL is about 4000 nM and

for Pfeiffer the IC50 is about 20 nM after 6 days of treatment

Experimental Protocol • Data was recorded after three days and after six

days• For the day three plate, in a costar 96 clear flat cell

culture plate, 5,000 Pfeiffer cells were plated in 16 wells and 2,500 RL cells were plated in 16 wells– the difference in cell number was due to the fact that RL

grows faster than Pfeiffer• A serial dilution of the drug GSK 126 was made

– For two experiments the drug concentrations were 20,000, 40,000, 800, 160, 32, 6.4 and 1.28 nm

– For the next two experiments the drug concentrations were 100,000, 10,000, 1000, 100, 10, 1 and .1 nM

• 50 uL of each of the concentrations were added to two wells of RL cells and 2 wells of Pfeiffer cells allowing for two trials at each concentration 7

Experimental Protocol • For the day six plate, 2,500 Pfeiffer cells were plated in 16 wells

and 1,250 RL cells were plated in 16 wells, this was done because the cells would become too confluent if they were not diluted for the day 6 plate

• 50 uL of each of the serially diluted concentrations was once again added to two wells of cells on the plate

• Along the edges of the day three and day six plates, 100 uL of media was added to prevent evaporation of the cells in the plate

• These plates were then put in the incubator• On day three, freshly prepared drug solutions were added to the

day six plate because the drug does not last six days and the cells would be too confluent– To deal with this problem, 50 uL of cells, drug and media was

taken out of each well and 50 uL of the fresh drug dilutions was added

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Experimental Protocol • After either three or six days, the plate was taken out and

to measure cell number• The CellTiter-Glo Luminescent Cell Viability Assay was

used to measure the cell number in each of the wells• Once the reagent was prepared, 100 uL was added to

each of the wells with cells to measure the cell number• The luminescence was then measured• The CellTiter-Glo reagent works by binding to the ATP in

cell and measuring the amount of ATP, thereby indicating metabolically active and alive cells

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Effect of GSK 126 on RL cells after 3 days of treatment

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Effect of GSK 126 on Pfeiffer cells after 3 days of treatment

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Effect of GSK 126 on RL cells after 3 days of treatment

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Effect of GSK 126 on Pfeiffer cells after 3 days of treatment

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• It can be seen that with the RL cells, the cell number does not change too much and remains pretty constant as the amount of drug treatment increases indicating that the RL cells are not sensitive to the drug. On the other hand there is a decrease in the number of Pfeiffer cells as the amount of drug treatment increases indicating that the Pfeiffer cells are sensitive to the drug.

• However we noticed that the cell number was not plateauing off as we expected, indicating we needed to use higher drug concentrations on the cells. This is what we did for experiment 6.

Experiment 3 Data: 6/20 Day 3

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Effect of GSK 126 on RL cells after 3 days of treatment

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It can be seen that with the RL cells, the cell number does not change too much and remains pretty constant as the amount of drug treatment increases indicating that the RL cells are not sensitive to the drug. On the other hand there is a decrease in the number of Pfeiffer cells as the amount of drug treatment increases indicating that the Pfeiffer cells are sensitive to the drug.

Experiment 3 Data: 6/23 Day 6

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Effect of GSK 126 on RL cells after 6 days of

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Effect of GSK 126 on Pfeiffer cells after 6 days of treatment

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It can be seen that with the RL cells, the cell number does not change too much and remains pretty constant as the amount of drug treatment increases indicating that the RL cells are not sensitive to the drug. On the other hand there is a decrease in the number of Pfeiffer cells as the amount of drug treatment increases indicating that the Pfeiffer cells are sensitive to the drug. The IC50 is about 10 nM

Conclusion and Future Plans• Our IC50 was about 10 nM for the Pfeiffer cell line based on the six day

treatment• This agrees with the published paper (McCabe et al “EZH2 inhibition as

a therapeutic strategy for lymphoma with EZH2-activating mutations”)• We can conclude that GSK 126 can be effective in inhibiting EZH2 over

activity in the Pfeiffer cell line, which gives a clinical therapeutic implication

• It is important to note however that it cannot be said that there is a direct relationship between GSK 126 sensitivity and the presence of an activating mutation in EZH2

• Previously published data suggests that both cell lines are sensitive to the drug in terms of H3K27 methylation, but differ in sensitivity in terms of cell growth

• The next step in analyzing these two cell lines would be to find out why RL and Pfeiffer cells respond differently to GSK 126. For example one could look into the gene expression of the cells in response to GSK 126 and see if it differs in RL and Pfeiffer cells

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Acknowledgements

• Prakash Rao• Melissa Duarte• Eilene Duberow

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PCR Background• Basics:

– DNA has all the genetic information, RNA is required for protein synthesis– RNA is transcribed from DNA when protein synthesis is required– The protein is then translated from RNA by the ribosome– Proteins consist of long chains of amino acids connected by peptide bonds

• PCR stands for polymerase chain reaction• PCR is a molecular biology technique for exponential synthesis of many copies of a

sequence of DNA• The chain reaction refers to repeated cycles of complementary strange synthesis

– The products of one cycle are templates for the next cycle• PCR yields semi quantitative data while QPCR yields highly quantitative data• The initial starting quantity of RNA is needed to understand how genotype affects

phenotype: this is how PCR is used• cDNA is complementary DNA: it is required as a starting template because the enzyme

used to synthesize copies is a DNA polymerase• So if trying to analyze RNA levels, the RNA must be reverse transcribed to cDNA so it can

be used in the PCR reaction, gene expression data is a reflection of starting RNA transcripts so important that CDNA is proportional to starting RNA

• But if analyzing DNA don’t have to RT

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PCR• PCR reaction happens in 3 steps: 1) denaturation 2) annealing 3) extension• 1) denaturation: splitting on DNA• 2)annealing: after DNA splits, the reaction is cooled and primers anneal to the

separated strands (bind to ends)– Primers anneal because that is the initial point for the DNA polymerase to

bind to the single DNA strand

• 3) once the primers have annealed, extension begins, after successful extension there is synthesis of a new strand of DNA known as the amplicon which is shorter

• These three main steps are repeated over and over at different temperatures, all 3 steps are one cycle

• 3 PCR phases– 1) exponential phase: doubling od amplicon occurs every cycle– 2) linear phase – rate of amplification slows as reagents are consumed

(nucleotides and primers)– 3) plateau phase – all reagents exhausted at this point

• Basically PCR amplifies specific DNA sequence, you pick the sequence – one part to be amplified and one non amplifiable part as a negative control, specific primers for that sequence

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QPCR• Qpcr determines amount of PCR product formed in an end point

PCR reaction, the product is visualized after the reactions complete

• In qpcr the amount of PCR product product (amplified DNA) formed is continuously monitored

• Fluorogenic dyes are used to measure the amplification, the due binds to double stranded products resulting in fluorescent signal

• You would expect a curve cycles on x, and fluorescent on y, indicating amplification

• Melt curve: want one peak for when one amplicon melts, to make sure you are amplifying something real and not just background– It makes the DNA melt and split apart so loses

fluorescence , and should only have one temperature since each DNA fragment is unique should have one peak should have only amplified one thing

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QPCR data

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QPCR for DNA Sample 1

Primer 1Primer 2

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QPCR for DNA Sample 2

Primer 1Primer 2

Amount of DNA

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More DNA takes less cycles because already starting out with so much DNA takes less time and cycles to amplify and get the threshold level