Kalsi Heimdal Background What is epigenetics What is an epigenetic modification How could it help with cancer therapy 2 Acute Myeloid Leukemia AML 3 Most common type of acute leukemia ID: 916238
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Slide1
New Approaches to Treating Cancer: Epigenetic Modifiers Can Help Leukemia Cells to Respond to Therapy
Kalsi
Heimdal
Slide2BackgroundWhat is epigenetics?What is an epigenetic modification?How could it help with cancer therapy?
2
Slide3Acute Myeloid Leukemia (AML)
3
Most common type of acute leukemia.
Approximately 8 subtypes.
Associated with specific, recurrent chromosome translocations, like the MLL
gene (known as a driver of AML).
All-trans-retinoic-acid (ATRA) therapy is a
successful, more attractive
differentiation therapy used to treat APL, but is ineffective for other AML subtypes
Slide4Hypotheses
Specific genes must be activated or inhibited in AML for differentiation therapy to work
AML with different genetic alterations may respond differently to specific epigenetic inhibitors.
4
Slide5Cell lines Inhibitors
MV4;11
Has the MLL
translocation
THP-1
Has the MLL
translocation
U937
Does not have the MLL translocation
5
CI-994
General histone deacetylase inhibitor
TCP
H
istone demethylase inhibitor
When possible, TPA was used as a reference for differentiation
Slide6Overview of Experiments Performed
Proliferation Assay
Direct measurement of cell proliferation.
MTT Assay
Measurement of cell
metabolic activity
.
FACS Analysis
Testing for the presence of specific cell markers.
6
Gene Expression Analysis
Analyzing any changes in gene expression of genes
involved
in AML.
Cytospin
Analysis
Qualitatively
assessing morphological changes of
differentiation.
Slide7Proliferation Assay
Cells were distributed in a 6-well plate.
Plate put in 37
o
C incubator after treatment with drugs.
Cells were counted 4 times over a 1 week period.
7
Slide8MTT Assay
Cells were distributed in 4 96-well plates.
Plate put in the incubator after treatment.
Plates were read
using
VERSAmax
Microplate Reader to assess cell metabolic activity
8
Slide9FACS Analysis
Cells were distributed in a 6-well
plate (same as proliferation assay setup).
Plate put in the
incubator for 4 days after
treatment.
Cells were stained at the 4
th
day with appropriate antibodies.
Samples were sent to the Flow Cytometry core at the University of North Dakota for analysis.
9
Slide10Gene Expression Analysis
Cells were distributed in
a 6-well plate.
Plate put in the incubator after
treatment for 4 days.
Performed RNA extraction, then cDNA synthesis
Performed RT-PCR to analyze
any
changes in gene expression of genes
associated with AML
.
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Slide11Cytospin Analysis
Cells were distributed in a 6-well plate.
Plate put in the incubator after treatment for 4 days.
At the 4
th day, cells were loaded into
cytospin
funnel attached to a slide, and put into the cytocentrifuge.
Cells were fixed using methanol, and stained using 3-Step
Stain Set
11
MV4;11 Untreated
Slide12U937 ResultsFACS Analysis
12
Gene Expression Analysis
Slide13THP-1 ResultsFACS Analysis
13
Gene Expression Analysis
Slide14MV4;11 ResultsFACS Analysis
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Gene Expression Analysis
To be performed
Slide15Cytospin Analysis—Results
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MV4;11 A+C THP-1 A+T
Slide16Conclusion
These experiments suggest epigenetic inhibitors may increase sensitivity to differentiation therapy, but that the response may still be dependent on the specific genetic alteration driving the AML.
Our studies suggest that in
MLL
-driven leukemia, the
MLL
fusion
oncoprotein
may override genetic manipulation resulting from treatment with epigenetic inhibitors, preventing differentiation by ATRA.
16
Slide17Future Directions
RT-PCR for gene expression analysis of the
MEIS1
gene
FACS analysis using CD34 and C-KIT antibodies
17
Slide18Thank you!
FACS analysis was performed by the North Dakota Flow Cytometry and Cell Sorting Core (ND-FCCS)
by Steven Adkins at
the University of North Dakota.
Research supported by an Institutional Development Award (
IDeA
) from the National Institute of General Medical Sciences of the National Institute of Health under grant number P20GM103442
.
Dr.
Super
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Slide19Questions?
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