DEVELOPMENT OF A MOUSE GENETIC MODEL OF WALDENSTROM’S MACROGLOBULINEMIA
Background: Recently Whole Genome Sequencing (WGS) was performed on tumor cells from patients with Waldenstrom’s Macroglobulinemia (WM), an effort co-funded by the International Waldenstrom’s Macroglobulinemia Foundation (IWMF). Analysis of the WGS revealed that in the 90% of the WM patients there exists the same genetic mutation in the gene encoding for a protein named MyD88. This single-point mutation, identified as L265P, in the MyD88 gene caused a different amino acid to be used to construct the MyD88 protein. Although the L265P mutation is highly recurrent and generates a protein that is more active than the wild type form in in vitro studies (cell lines), the role that this mutation plays in vivo (transgenic mice) in the initiation or progression of WM has yet to be determined.
In order to understand how the L265P mutation affects the development of WM two main research models can be used. One model involves the use of competent cell lines which are initially isolated from patients with WM and established for long term culture in ex vivo conditions. As a part of the initial efforts WM cell lines which contained this mutation were used. Treatment of these cell lines with agents that block the function of MyD88 protein or some of its downstream targets was associated with apoptosis or death of the WM cells, underscoring the pathogenetic role of this protein and its potential role as novel therapeutic target. The other model involves the use of transgenic mice. Below is an outline of the Specific Aims that Dr. Carrasco will follow in an effort for developing a mouse transgenic model that contains the MyD88/L265P mutation so prevalent in WM patients.
Specific Aim 1: Generate conditional MyD88 (L265P) mice
A “piece” of genetic material containing the MyD88 gene with the L265P mutation will be incorporated into the mouse genome. This piece of genetic material will also contain a biological “switch” that will not allow the MyD88 gene containing the L265P mutation to be expressed until a certain event occurs, see below. A similar process will be followed using genetic material containing the “wild type” MyD88 gene without the L265P mutation, to act as a control. This is done to assure that the process is not the cause of any later reactions in the development of the mice and any differences are due to the L265P mutation. These mice will be bred and progeny will be evaluated to assure that they contain the changes described above.
Specific Aim 2: Generate MyD88/AID compound mice
Using the mice developed in Specific Aim 1 they will be cross bred with a second line of mice that will activate the “switch”. This second line of mice has a construct that encodes” Cre”, a protein normally not expressed in the mouse and that when bred with mice generated in Specific Aim 1 will turn the switch ON and activate expression of MyD88 containing the L265P mutation. As in Specific Aim 1, these mice will be bred and the progeny will be analyzed to assure that the cross breeding was successful and the bred mice contain genetic information from both line of mice. This switch will be activated only in B-cells that have recognized a pathogen because the construct that encodes “Cre” is regulated by genetic material that control the expression of the Activation-Induced Cytidine Deaminase (AID) protein that is normally expressed at this stage of B-cell development. It is at this time of the B-cell development, in today’s understanding, that WM cells originate. Note that MyD88/L265P expression from birth can be a lethal event, so another reason to delay its activity by generating conditional mice.
Specific Aim 3: Characterize MyD88/AID compound mice
With the mouse “model” of WM now in place, the mice will be monitored to watch for the development of the symptoms and biological values that we see in patients with WM. Periodic Clinical Exams will be performed to assess for stress, ill health and tumors as well as analysis of peripheral blood smears. Those that do show evidence of disease development will be euthanized to perform more detailed analysis. Also, a set of Serial Studies will be performed to evaluate for tumor progression. In these studies a subset of the mice will be periodically sacrificed and an extensive set of analyses will be performed looking at bone marrow as well as many of the organs to assess disease involvement. Included in this evaluation will, of course, be the evaluation of any immunoglobulin production to assess for clonality, looking for the IgM “spike”. Additionally RNA will be examined to help assess the activity of pathways within the B-cell as the disease progresses. Particular attention will be paid to those proteins and other molecules in the B-cell associated with the MyD88 and its down stream pathways. An Endpoint Study will assess how long the mice with the mutation live in comparison to the Control Mice described in Specific Aim 1.
Specific Aim 4: Translational use of the MyD88/AIM compound mice
Now that the mice that contain the MyD88/L265P mutation are in place and display the clinico-pathological features that we see in WM patients, inhibitors to the proteins and molecules associated with MyD88 and related pathways will be evaluated. There are already in place some drugs that work in this area from other autoimmune and inflammatory diseases and they will be tried initially. Once again effects on survival will be collected and a comparison will be made between those that do receive treatment and those that do not.
This is a planned three year effort that takes into consideration the normal life expectancy of mice (two years). It takes time for the mice to breed and successfully produce progeny that contain the effects that Dr. Carrasco will introduce; you know how Mother Nature works. This will encompass the first year of the project. Then once the mice that contain the modifications are available it will take time for the WM to evolve and to execute the evaluation of therapeutic agents, this will be done in years 2 and 3. Once the genetic mouse model of WM is established and it’s preclinical applicability is assessed it will be available to other researchers and clinicians working to find a cure for this dreaded disease.