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Is RpS6 activation important for leukemia cell progression?

Peña Martínez, Pablo E. (2013) MOBN19 20131
Degree Projects in Molecular Biology
Abstract
Popular science summary:

Is RpS6 activation important for leukemia cell progression?

Acute Myeloid Leukemia (AML) is the most common acute blood cancer in adults. It is characterized by a rapid increase of leukemic cells in the bone marrow, compromising the
development of new blood cells. Despite improvement in treatments and advancements in
supportive care, the overall 5-years survival of AML is around 30%, showing the bad prognosis that this disease has.

AML relapses in a high percentage of cases after an initial response to the treatment, and usually this relapsed AML is harder to treat than the primary AML. The reason why the disease reappears is thought to be due to the presence of the so-called leukemic stem cells (LSCs),... (More)
Popular science summary:

Is RpS6 activation important for leukemia cell progression?

Acute Myeloid Leukemia (AML) is the most common acute blood cancer in adults. It is characterized by a rapid increase of leukemic cells in the bone marrow, compromising the
development of new blood cells. Despite improvement in treatments and advancements in
supportive care, the overall 5-years survival of AML is around 30%, showing the bad prognosis that this disease has.

AML relapses in a high percentage of cases after an initial response to the treatment, and usually this relapsed AML is harder to treat than the primary AML. The reason why the disease reappears is thought to be due to the presence of the so-called leukemic stem cells (LSCs), which are present in a quiescent state and therefore remain immune to the chemotherapy that targets dividing cells. In order to avoid disease relapse and cure AML, the LSCs need to be eradicated.

How to target the LSCs without affecting the normal hematopoietic stem cells responsible for normal blood cell production is one of the main challenges for developing more effective treatments. Such treatments should ideally be directed to molecular targets in the LSCs that are essential for their survival. A way to find such targets is by performing a short hairpin RNA (shRNA) screening, which is a method to suppress genes and thereby to get information on whether they are important for particular cells, for example LSCs. In one of such screens, it was found that Casein Kinase 1α (Csnk1a1) is crucial for LSCs. Further studies on Csnk1a1 pointed out the Ribosomal Protein S6 (RpS6) as one downstream mediator of Csnk1a1 signaling essential for LSCs. RpS6 can be phosphorylated directly by Csnk1a1 as a response to growth and proliferation stimuli, but how this phosphorylation affects leukemic cells has not previously been studied. The aim of this project was to determine whether the phosphorylation of RpS6 is important for leukemic cells.

The first step to determine the role of RpS6 phosphorylation was the creation of viral vectors. One of the viral vectors was used to knock-down the endogenous RpS6 in the leukemic cells, while the other vector was used to express an RpS6 gene whose phosphorylation status we could modify. This RpS6 gene was designed so it could either be expressed as a wild-type protein or as a mutated protein that cannot be phosphorylated. With these two RpS6 variants, a rescue experiment was performed, in which we could study whether RpS6 phosphorylation is critical for the proliferation of leukemic cells.

We found that RpS6 expression rescued the proliferation defect induced by knockdown of the endogenous RpS6; i.e. cells began to proliferate more when the exogenous RpS6 was expressed. This effect seemed to be independent of the phosphorylation status of RpS6, as no difference could be seen between the wild-type and the phosphorylation deficient mutant of RpS6. Therefore, our data suggest that RpS6 phosphorylation is dispensable for the proliferation of leukemic cells in a short-time culture. Additional studies with a constitutively active mutant of RpS6 in the context of Csnk1a1 knock-down will be performed to further explore whether RpS6 phosphorylation is important for leukemia cell progression.

Master’s Degree Project in Molecular Genetics 45 credits
Supervisor: Marcus Järås, PhD
Department of Clinical Genetics, BMC, Lund University (Less)
Please use this url to cite or link to this publication:
author
Peña Martínez, Pablo E.
supervisor
organization
course
MOBN19 20131
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
4053968
date added to LUP
2013-09-19 11:40:48
date last changed
2013-09-19 11:40:48
@misc{4053968,
  abstract     = {Popular science summary:

Is RpS6 activation important for leukemia cell progression?

Acute Myeloid Leukemia (AML) is the most common acute blood cancer in adults. It is characterized by a rapid increase of leukemic cells in the bone marrow, compromising the
development of new blood cells. Despite improvement in treatments and advancements in
supportive care, the overall 5-years survival of AML is around 30%, showing the bad prognosis that this disease has.

AML relapses in a high percentage of cases after an initial response to the treatment, and usually this relapsed AML is harder to treat than the primary AML. The reason why the disease reappears is thought to be due to the presence of the so-called leukemic stem cells (LSCs), which are present in a quiescent state and therefore remain immune to the chemotherapy that targets dividing cells. In order to avoid disease relapse and cure AML, the LSCs need to be eradicated.

How to target the LSCs without affecting the normal hematopoietic stem cells responsible for normal blood cell production is one of the main challenges for developing more effective treatments. Such treatments should ideally be directed to molecular targets in the LSCs that are essential for their survival. A way to find such targets is by performing a short hairpin RNA (shRNA) screening, which is a method to suppress genes and thereby to get information on whether they are important for particular cells, for example LSCs. In one of such screens, it was found that Casein Kinase 1α (Csnk1a1) is crucial for LSCs. Further studies on Csnk1a1 pointed out the Ribosomal Protein S6 (RpS6) as one downstream mediator of Csnk1a1 signaling essential for LSCs. RpS6 can be phosphorylated directly by Csnk1a1 as a response to growth and proliferation stimuli, but how this phosphorylation affects leukemic cells has not previously been studied. The aim of this project was to determine whether the phosphorylation of RpS6 is important for leukemic cells.

The first step to determine the role of RpS6 phosphorylation was the creation of viral vectors. One of the viral vectors was used to knock-down the endogenous RpS6 in the leukemic cells, while the other vector was used to express an RpS6 gene whose phosphorylation status we could modify. This RpS6 gene was designed so it could either be expressed as a wild-type protein or as a mutated protein that cannot be phosphorylated. With these two RpS6 variants, a rescue experiment was performed, in which we could study whether RpS6 phosphorylation is critical for the proliferation of leukemic cells.

We found that RpS6 expression rescued the proliferation defect induced by knockdown of the endogenous RpS6; i.e. cells began to proliferate more when the exogenous RpS6 was expressed. This effect seemed to be independent of the phosphorylation status of RpS6, as no difference could be seen between the wild-type and the phosphorylation deficient mutant of RpS6. Therefore, our data suggest that RpS6 phosphorylation is dispensable for the proliferation of leukemic cells in a short-time culture. Additional studies with a constitutively active mutant of RpS6 in the context of Csnk1a1 knock-down will be performed to further explore whether RpS6 phosphorylation is important for leukemia cell progression.

Master’s Degree Project in Molecular Genetics 45 credits
Supervisor: Marcus Järås, PhD
Department of Clinical Genetics, BMC, Lund University},
  author       = {Peña Martínez, Pablo E.},
  language     = {eng},
  note         = {Student Paper},
  title        = {Is RpS6 activation important for leukemia cell progression?},
  year         = {2013},
}