Senior
Project Proposal
Angela
Hemesath
February 23
2014
- Title of Project:
Targeting Multiple Myeloma’s
Dependence on IRF4 by Antisense Oligonucleotide Drugs
- Statement of Purpose:
Multiple Myeloma
is a cancer of plasma cells (the antibody-producing of white blood cell). As
the cancer progresses, the malignant plasma cells accumulate in the bone
marrow, crowding out healthy cells (“Diseases and Conditions Multiple
Myeloma”). Multiple Myeloma may cause weight loss, bone fractures, anemia, or
bone cell deficiency (Courseault,
2014).
Although Multiple Myeloma is a relatively rare disease, about 25, 000 new cases
are reported in the US per year (Cancer.org). Though no cure exists for
multiple Myeloma (Davis, 2014), the cancer has been researched genetically in
the field of individualized medicine. Studies have shown that many cases of
Multiple Myeloma involve the translocation of the MYC gene involving an Ig
locus (Dib, 2014). The MYC gene expresses a protein that is in cell cycle
progression and cell proliferation. The dysregulation of MYC (when MYC is
expressed in excess) is associated with tumors (“Summaries for MYC Gene”).
Studies have
suggested that Multiple Myeloma cancer cells are dependent on the transcription
factor (a protein that binds to specific genes to control the formation of mRNA
from DNA) IRF4. According to this research, the inhibition of IRF4 may be toxic
to Myeloma cell lines (Shaffer et al., 2008).
ISIS
Biotechnology in Carlsbad, California has produced a drug that uses antisense
oligonucleotides (ASO’s) to block IRF4 gene expression by binding IRF4 mRNA and
stopping translation, and killing part of the Myeloma population. This drug’s
effectiveness have not been analyzed in a variety of different Myeloma cell
types.
Experimentally, we hope to
determine which cell lines are sensitive or resistant to IRF4 ASO’s. Using
these results, we will determine if there is a genetic correlation with drug
sensitivity. Through the experiment, an understanding of Multiple Myeloma’s
dependence on IRF4 will be obtained, which may provide an opportunity for
individualized medicine.
- Background:
I have been an intern at the Mayo
Clinic in Scottsdale for a year, and have been learning about Multiple Myeloma
throughout my internship. Biology classes prepared me for the knowledge and
critical thinking that I would require. Having already had an interest in
biology, I was further attracted to the topic of genetics by the class and
subsequently immersed in the world of genetics by my internship. Because of
this, genetic engineering and the idea of predicting and treating disease
through genetics fascinates me.
- Prior Research:
Multiple Myeloma is a cancer of
plasma cells (Davis). These plasma cells accumulate in the bone marrow, causing
bone pain, anemia, and other side effects (“Diseases and Conditions”). Multiple
Myeloma is considered to be incurable.
The MYC translocation (the locus rearrangement
of genes around MYC) was the first translocation found in cancer. However, the
expression of MYC cannot occur without the presence of IRF4 (Shaffer et al.,
2008).Since the MYC gene transcription factor pushes cell proliferation, its
dysregulation often results in tumors or cancers. MYC translocations may drive MGUS into Multiple
Myeloma. MGUS-- Monoclonal gammopathy of undetermined significance—is an
abnormal protein present in the blood (“Monoclonal Gammopathy of Undetermined
Significance (MGUS)”). MGUS may result in a premalignant tumor present in 4% of
adults above the age of 50. Eventually, MGUS may become Multiple Myeloma in 1%
of people. Out of those 1% of people, 50% showed a translocation of the MYC
gene (a regulator gene that codes for transcription factor). This results in
the dysregulation of the MYC gene, which is associated with the initiation of
Multiple Myeloma (Bergsagel and Kuehl, 2002).
MYC is a direct target of IRF4 in Myeloma
cells, and IRF4 is a direct target of MYC. According to a study in 2008, Myeloma
in which IRF4 production was blocked were killed, with non-Myeloma unaffected.
This occurred without genetic abnormalities in IRF4 locus. The study concluded
that Myeloma is “addicted” to IRF4, regardless of subtype (Shaffer et al.,
2008)
ASOs
(antisense oligonucleotides) bind with complimentary mRNA, blocking the
expression of a protein (in this case, with the hope to stop formation of IRF4).
Research by ISIS (the biotech company in Carlsbad, CA) showed that drugs which
utilized the ASO’s worked in certain cell lines (KMS11 should show a marked
decrease in metabolic activity, but U266 should not since it does not have a
dependency on MYC), but not for all Myelomas.
- Significance:
Multiple Myeloma is present is more
men than women, and more African-Americans than Caucasians. Only 30-40% of
patients have a complete response to initial treatment, with most only
surviving 3-4 years (Bergsagel). Multiple Myeloma is currently treated in such
a way that all plasma cells, benign or malignant, are destroyed in the process.
This is a very general form of treatment, which does not work for most, and
often leaves patients vulnerable to toxicity.
A wide spectrum of patient outcomes
has been observed, ranging from no improvement to remission. This leaves
patients at the mercy of trail-and-error treatments that may or may not work
depending on the patient’s genes. And, given that Myeloma has 5-10 genetic groupings,
patients may have very different treatment experiences.
If the sensitivity of Multiple Myeloma
to these ASO’s can be predicted by the genetic mutations driving multiple
myeloma, then ASO’s can be used to efficiently target malignant tumors successfully
in the appropriate patients, minimizing costly and/or painfully non-productive
therapy.
- Description:
My research will be done in the
Bergsagel Lab at the Mayo Clinic. The work will be at the cellular as well as
molecular level. My research will focus on the effect of different genetic
backgrounds and the efficacy of targeting IRF4. I will be several ASOs of
increasing dosages on a number of cell types in order to determine the
effectiveness of the drug, as well as which cell lines are resistant.
- Methodology:
There will be 2 stages of the
experiment.
Stage one will be cell line
treatment, which will include maintainting the cell lines and then preparing
them for the experiment. The cells must be diluted to a concentration of around
20,000-50,000 cells/ml. These will then
be seeded into a 96 well plate with the drugs containing the ASO’s at differing
concentrations that may range from 80nM-10uM.
In the
second step, we will analyze each stage of the response. We hope to test the
effect of the ASO’s at each stage of expression. Through different assays such
as cell count, viability and metabolic activity assays. This way, we hope to
show experimentally the effect of the ASO’s on the proliferation of the cells,
the mRNA inhibition, and, ultimately, synthesis of the IRF4 and MYC protein.
- Potential Problems:
Bergsagel
lab has a wide variety of cell lines that would be beneficial to test with the
ASO’s. Though there may be some anticipated difficulty in replicating ISIS
Biotechnology’s experiments, we can collaborate easily to make sure that the
experiment runs smoothly and correctly.
The cell
lines are very good representations of the tumors from which they are derived—that
is, they mimic the gene expression very closely. However, there are some differences
between the behavior of the cell lines and the tumors. However, the cell lines
are a good first step toward more advanced studies in animal models as well as
clinical trials.
- Bibliography:
A, Jenny. “Myc in Myeloma – Why it’s
a Prevalent and Exciting Target.” Myelomacrowd.org.
Myeloma Crowd, 13 March 2014. Web. 16 Dec. 2014.
Bergsagel,
Leif P., and W. Michael Kuehl. “Multiple Myeloma: Evolving Genetic Events and Host Interactions.”
Macmillan Magazines Ltd March 2002: 175- 187.
Print.
Courseault, Jacques. “Long-term Side Effects of
Multiple Meyloma.” Livestrong.com.
Livestrong, 22 June 2010. Web. 16 Dec. 2014.
Davis, Charles P. “Multiple Myeloma.” Medicine.net.
Medicine.net, 17 Sept. 2013. Web. 16 Dec.
2014.
“Characterization of MYC
Translocations in Multiple Myeloma Cell Lines.” Nih.com. NCBI, 3 Sep.
2009. Web. 16 Dec. 2014.
“Diseases
and Conditions Multiple Myeloma.” Mayoclinic.org. Mayo Clinic, n.d. Web. 9 Oct. 2014.
Martín-Subero
JI, Odero, Hernandez R, Cigudosa JC, Agirre X, Saez B, Sanz- García E, Ardanaz MT, Novo FJ, Gascoyne RD, Calasanz MJ, Siebert R. “Amplification of
IGH/MYC fusion in clinically aggressive IGH/BCL2-positive germinal center B-cell lymphomas.”
Nih.gov. NCBI, Aug. 2005. Web. 16 Dec.
2014.
“Monoclonal
Gammopathy of Undetermined Significance.” Mayoclinic.org. Mayo Clinic, 17 May 2013. Web. 16
Dec. 2014.
“Multiple Myeloma.” Cancer.org. American Cancer
Society, 16 June 2014. Web. 16 Dec. 2014.
Shaffer, Arthur L., Emre, N.C. Tolga, Lamy,
Laurence, etc, 2008, “IRF4 Addiction in Multiple
Myeloma.” Nature, v. 454, p. 226-231.
“Summaries for MYC Gene.” Genecards.org. Genecards. N.d. Web. 16 Dec. 2014.
“Summaries for MYC Gene.” Genecards.org. Genecards. N.d. Web. 16 Dec. 2014.
“Transcription Factor.”
Britainica.com. Britainica. N.d. Web. 16 Dec. 2014.
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