This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.

Further Directions

There is still much to be learned about the genetic basis of Juvenile Myelomonocytic Leukemia. Even though researchers have identified genes (such as KRAS) that is found to be mutated in JMML patients (and mouse models of JMML), many of the pathways, in particular downstream targets that cause the JMML phenotype are still unknown. Below is my proposed experiments to learn more about JMML and KRAS.

Proposed Experiment One

The first experiment that I propose is based on one important feature of JMML.JMML is caused by spontaneous growth of colony forming units-granulocyte/macrophage (CFU-GM). JMML bone marrow cells are shown to be hypersensitive to Granulocyte macrophage colony-stimulating factor (GM-CSF).

The above diagram shows the pathways in which JMML is believed to act through. The hypersensitivity to GM-CSF in JMML is an attribute that necessarily involves the GM-CSF receptors. My first experiment is to test for any abnormalities in the GM-CSF receptors (GM-CSFR) in JMML bone marrow cells. To do this, I suggest using PCR sequencing to screen for genetic abnormalities in the GM-CSFR coding region in human JMML patients. If there are any mutations in these genes (GM-CSFR consist of two subunits, we can test it through the following experiment. The experiment below is based on the assumption that an abnormal mutation has been found in the beta subunit of the GM-CSFR.

Suppose a mutation in the beta subunit is found in the receptor. To compare it's effects to the wild type, we can create a mouse bone marrow cell line that has the wild-type human alpha subunit coding region. After that, we then use that existing cell line to create two more lines - one containing the novel beta mutation found in the JMML patients and the other containing just the wild type human beta subunit. When the two cell lines are created, we can expose them to GM-CSF and see if there is hypersensitivity in the novel mutation compared to the wild type. This could possibly be observed through more growth in the cell line with the novel mutation if there is hypersensitivity.

Proposed Experiment Two

The next question I want to answer through my proposed research is if KRAS can initiate JMML in human bone marrow cells. Previous research has shown that mice transplanted with bone marrow containing the KRAS JMML mutation (KRAS G12D) can initiate JMML-like symtomps. This is important because even though a mutated KRAS is found in JMML bone marrow cells, we do not know that if it is some other factor that causes the cells to behave abnormally and then the mutation occurs, or if the mutation is what causes the cell to become a JMML cell in humans.

I propose a simple experiment to test this hypothesis. To see if KRAS can initiate JMML, I propose to simply create a trasgenic human bone marrow cell line containing the KRAS mutation in JMML patients. I would expose the bone marrow cells to most of the growth factors that it would normally be exposed to in it's natural state. After that, we can observe the cell culture to see if any JMML like attributes occur after a period of growth. If the cells begin to exhibit JMML-like phenotypes such as hypersentivity to GM-CSF or more white blood cells than the control, it would generate evidence for KRAS being able to initiate JMML in humans.

Proposed Experiment Three

My last experiment simply seeks to identify more of the downstream targets of KRAS in JMML patients. To do this, I propose a traditional microarray experiment measuring gene expression in KRAS-mutated JMML patients. Any genes that is found to be overexpressed or underexpressed can then be tested through knock-out or overexpression experiments in a murine model. Learning more about the downstream pathways could lead to advances in drug discovery to help JMML patients.

Recent Developments

Interestingly, recent research in UC - San Francisco has led to the discovery of a drug that inhibits the JMML phenotype in KRAS-mutated mice. The drug functions by inhibiting the MEK pathway, one of the downstream targets of KRAS. Below is a quote from one of the researchers in the study.

“The most striking aspect of our findings was that the treatment helped the mice not by making the cancer cells go away, but by forcing them to act like normal cells, despite their mutation.” Natalya Lyubynska, MD.

This significant discovery shows that by further exploring the KRAS downstream pathways, we can learn more about the disease to help JMML patients.

The above figure shows the effectiveness of the new drug in mice in inhibiting JMML phenotype.

References

Oncogenic Kras-induced leukemogeneis: hematopoietic stem cells as the initial target and lineage-specific progenitors as the potential targets for final leukemic transformation
http://bloodjournal.hematologylibrary.org/content/113/6/1304.abstract

UCSF Researchers Identify Promising New Treatment for Childhood Leukemia
http://www.ucsf.edu/news/2011/03/9631/ucsf-researchers-identify-promising-new-treatment-childhood-leukemia

Targeting RAS signaling pathways in juvenile myelomonocytic leukemia.
http://www.ncbi.nlm.nih.gov/pubmed/17584027

RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML).
http://www.ncbi.nlm.nih.gov/pubmed/10049057

Successful treatment of juvenile myelomonocytic leukemia relapsing after stem cell transplantation using donor lymphocyte infusion
http://bloodjournal.hematologylibrary.org/content/101/5/1713.full