11% to completion
$30,445 / $268,000 goal
"Every race should be won … especially when it involves the life of a child"
— Jen & Mike, Greg's parents

Charles Keller

Scientific Director
Children's Cancer Therapy Development Institute

As a physician-scientist, my laboratory is devoted to the development of novel molecular therapies for advanced childhood muscle tumors associated with high morbidity or mortality. The long-term emphasis of my laboratory’s research is molecularly-targeted therapies to halt progression or induce regression for gross residual disease, metastatic disease and relapsed disease.  We recruit not only cancer biologists but also developmental biologists, muscle biologists and engineers to the investigation of these devastating soft tissue sarcomas. 

Summary

AMAZING!  - our thanks to the anonymous donor who is offering a 1:1 match up to $267,000 for this project!  

Recently, a teen developed an alveolar rhabdomyosarcoma (aRMS) with the typical Pax3:Foxo1 mutation found in this type of childhood cancer.  And yet there was another gene variation found… a second mutation.  maybe a cancer-causing mutation, maybe a bystander.  A key question is whether this mutation makes the cancer unique, or common?  Where does this child’s situation fit in with most other cases?  If this combination of mutations is unique, then are there other single-patient exceptional cases?  or instead, do manageable subtypes exist across the continuum of alveolar rhabdomyosarcoma cases – subgroups for whom customized treatments can be assigned?  Can we define a new standard of care for every subgroup of children battling aRMS?   

Why is this important?

This project is important because many children, teens and young adults with alveolar rhabdomyosarcoma need new treatment options.  When metastatic, the five-year survival rate is only 8%.  We want aRMS to be universally survivable --- with the least side effects and highest quality of life after treatment.    

 

Who will benefit?

Children, teens and young adults with alveolar rhabdomyosarcoma will benefit from new drug treatments, matched to a child’s tumor’s genetics, being introduced into clinical trials. 

Budget

This project in its basic form runs two years at $134,000 per year ($268,000 total).  The expenditure category breakdown is given below. It is supervised by Dr. Keller, and led first-hand by a postdoctoral fellow.  The project has both bioinformatics / computational biology and wetlab experiment studies.   If this crowdfunding project reaches its goal, our anonymous donor will match each dollar 1:1, doubling the funds available allowing us to hire an engineer to work alongside our postdoctoral fellow... doubling the speed and throughput towards our mission find the best treatment option for each group of children with alveolar rhabdomyosarcoma. 

Updates

11/30/2017 update

What is your Endotype?  Based on RNA expression of genes in aRMS, at least 6 subgroups (archeotypes) of aRMS exists (unpublished data).  For example, PI3K is over-expressed in many aRMS… which subgroup is this? what mouse model or what cell line best represents the PI3K subgroup?  What is the closest subgroup that does not express PI3K?  note:  this analysis does not yet have known cell lines added, but will. 

01/11/2018 update

Dear GEAR Supporters!

This week we have pursued multiple critical elements for the GEAR project! We have completed the exome sequencing that was initiated last week for tumor samples associated with the GEAR endotyping project. This week, we acquired a few RMS surgical biopsy and legacy gifts (after-life tissue for research) and have made progress on acquiring new critical samples; these can also be sequenced as part of the GEAR project. The next round of sequencing should take place in the coming 2-4 weeks, as samples arrive. My work this week on refining sequencing analysis scripts should make subsequent analysis faster and require less oversight.

We are have asked a good colleague whether additional cell line sequencing data can be shared with us in a collaboration. This will be key in the GEAR dendrogram to define endotype clusters in aRMS.

With the current available data, I will begin the process of developing the GEAR endotype dendrogram. Current code exists to develop such diagrams quickly, iteration will be based on addition of new datasets and exploration of varying aRMS-related gene sets. Next step, identifying aRMS endotype subgroups that may be predictive of therapy response.

02/22/2018 update

Dear GEAR supporters!

We have reached the first critical milestone in the GEAR project. In particular, we now have a robust working codebase for fast and accurate generation of endotype dendrograms. The code for the dendrograms has undergone several ease-of-use modifications to allow samples to be added with minimal work, and to add or remove genes from the DNA and RNA feature list, and to add or remove traits from the dendrogram key. This means new genes and features (such as clinical indicators, biomarkers, or other important clinical considerations) can be added or remove from the dendrogram with ease. The new result is that any new samples can be added the moment data is ready.

On the new sample front, my colleague Cora has shared some new data for DNA sequencing (currently running through our analysis pipeline), and she has grown some new RMS cell lines for DNA and RNA sequencing. These resources will be especially valuable as cell lines are some of the most robust experimental models available. Once we map the cell lines onto the current (and soon to expand) set of RMS samples, we will expand our understanding of endotypes in aRMS and have the right set of experimental models to identify promising new treatments for the endotype groups. With the support of the GEAR advocate group, we will soon sequence additional patient samples. We also have a new set of cell lines coming from the Children’s Oncology Group, which will further strengthen our database of RMS models.

We have an initial set of dendrogram-informed experiments coming up soon, so please stay tuned. The experiments will likely begin early next week. Round one is on its way!

3/23/2018 update

Dear GEAR Supporters,

Following up on finalization of the aRMS dendrogram, we have identified multiple preliminary aRMS endotypes now guiding experiments. Two of the endotypes represent lesser served and lesser known aRMS subtypes: the established but poorly understand Pax7:Foxo1 fusion aRMS, and an aRMS subtype with aberrant RET expression. These are in addition to the known Pax3:Foxo1 fusion subtype (which appears to have sub-classes soon to be explored), and a fusion-negative/atypical fusion subgroup. We have decided to first pursue the aberrant RET aRMS subgroup and the Pax7:Foxo1 fusion aRMS subgroup.

A key challenge is that while several primary tumor samples show elevated levels of RET, this behavior is not captured once cancer cells are grown in petri dishes. Thus, the past 2 weeks have been focused on transfection and confirmation of transfection of RET into aRMS cell models. Based on the GEAR dendrogram, we identified 4 cell models that could serve as representative of the RET subtype: Rh30, CF1X, U23674, and U31431. We selected these four models to serve as our experimental models for defining a promising treatment for aRMS with RET.

The results of RET transfection experiments were validated via western blot (protein quantification) experiments, which confirmed the modified cells were now expressing RET. This will allow us to perform drug screening and validation experiments to better understand the chemical space of this unanticipated aRMS endotype, including single drug and combination drug validation experiments. This experimental set will soon be underway. We also performed a preliminary western blot experiment exploring the effect of entinostat in RET-positive aRMS, which will be repeated soon with transfected cell lines.

Pax7:Foxo1 aRMS represents a challenging research topic, as there are presently only three published Pax7:Foxo1 positive cell models for research use: CW9019, RMZRC2, and HA-OH1. We currently have CW9019 in the lab, and we are accessing RMZRC2 through the originating research lab. The HA-OH1 cell line will require additional hunting, but if the sample exists in a research lab somewhere, we will get access to it. We also have access to the (world’s first) patient-derived xenograft model of Pax7:Foxo1, which we are attempting to establish into a cell model for research use. We will begin screening experiments on Pax7:Foxo1 shortly.

Thank you very much for your continued support, we will have more experimental data soon.

And in honor of National Puppy Day, I present the two adorable mascots for these two aRMS endotypes.

03/28/2018 update

Dear GEAR Supporters,

Today I have updates on the two endotypes we are pursuing for the GEAR project.

On the Pax7:Foxo1 aRMS endotype sub-project, we have an expected April 7/8 arrival date for the RMZRC2 cell line, so we will have two (potentially three) lines for this rare aRMS. We have still been unable to find a source for HA-OH1, though we have contacted the original authors and have exchanged emails. Hopefully they will be able to connect us with sources for the cell line soon.

On the RET-positive aRMS side, the next few weeks will be busy with experimentation. This week I have started experimentation on the two mouse cell lines. The cells were plated into initial experimental groups yesterday (plated in experimental and control triplicate, for a total of 12 concurrent experiments) and transfection is underway in 4 of the 12 today. Initial cell populations were staggered to ensure technical replicates were performed on different days. Tomorrow, the transfected cells will undergo single agent validation in 6 promising drugs, and combination validation in 5 combinations (based on the 6 promising drugs). The mouse cell line experiments will conclude approximately 1 week from today.

Due to the time requirement and technical difficulty of the planned experiments, I am staggering the cell lines to two per week. I will begin the human cell line experiments early next week (but more likely over the weekend).

Thank you very much for your continued support, we are working hard to complete experimental sets as quickly and accurately as possible!

04/04/2018 update

Dear GEAR Supporters,

Today I have updates on the two endotypes we are pursuing for the GEAR project.

There are currently no new updates on the Pax7:Foxo1 aRMS endotype sub-project, we are primarily waiting for arrival of the new cell line to begin screening and sequencing.

On the RET-positive aRMS side, 2 of three technical replicates of both mouse cell line experiments have been completed, with the third replicate starting in the next couple of days. The human experiments will also begin this week: sub-plating into experimental replicates took place this morning, and the first transfections will begin tomorrow.

Thank you very much for your continued support, we are working hard to complete experimental sets as quickly and accurately as possible!

04/11/18 update

Dear GEAR Supporters,

Today I have updates on the two endotypes we are pursuing for the GEAR project.

On the Pax7:Foxo1 aRMS endotype sub-project, we are waiting for arrival of the new cell line to begin screening and sequencing. This cell lines has been shipped from Italy and will arrive in the next day or two.

On the RET-positive aRMS side, all technical replicates of both mouse cell line experiments have been completed, and formal analysis of the mouse cell line data is underway. We are primarily looking for sensitivity of single agents and synergy of drug combinations. The human experiments are underway, with the first three technical replicates being completes this weekend. One of these has already been completed, and unfortunately will need to be performed again. The replacement replicate is in preparation.

Thank you very much for your continued support, we are working hard to complete experimental sets as quickly and accurately as possible!

Noah

04/18/18 update

Dear GEAR Supporters,

Today I have updates on the two endotypes we are pursuing for the GEAR project.

On the Pax7:Foxo1 aRMS endotype sub-project, the new cell line has arrived. The collaborators in Italy have sent a live culture, which can be somewhat difficult to do overseas. We are monitoring the culture now and sharing updates with the cell line’s creators to determine if the cell culture is recovering well. Our planning meeting this week outlined the next steps in agents to explore in PAX7 ARMS, so there will be more to come!

On the RET-positive aRMS side, all technical replicates of both mouse cell line experiments have been completed, and the formal analysis of the mouse cell line data is complete. Some time and work went into making the data as visually clean and appealing as possible. Thanks to the power of software, it’s (almost) an easy task.  Half of the experimental replicates for the human samples are now complete. I am currently analyzing the generated data to determine if the variation in the data is within acceptable limits. We will not take anything but the cleanest, most robust data to support our science here at cc-TDI.

Thank you very much for your continued support, GEAR heads! We are making great progress in the first of many ARMS endotypes!

04/25/2018 update

Dear GEAR Supporters,

Today I have updates on the two endotypes we are pursuing for the GEAR project.  On the Pax7:Foxo1 aRMS endotype sub-project, the new cell line has arrived. The live culture sent by our overseas collaborators has so far been showing less then desirable recovery. Our contact returns next week, and we will set up a shipment of a cryopreserved sample, which will be much easier to use to establish the cell line in our lab.

On the RET-positive aRMS side, I have been working through the human cell line replicates. One of the two cell lines may have higher than expected growth rates in the petri dishes, which I am concerned may be skewing experimental results. I’m re-running an experiment at a lower cell population to determine if the change in cell count helps clarify the results. The experiments for the other human cell line have not had any issues, and are proceeding as expected. Experiments should be finished in the next week or two.

Thank you very much for your continued support, GEAR heads!

Noah

05/02/2018 update

Dear GEAR Supporters,

Today I have updates on the two endotypes we are pursuing for the GEAR project.

On the Pax7:Foxo1 aRMS endotype sub-project, the cell line sent by our overseas collaborator has not recovered well. We are working to have a new cell line sent to us now. I will have further updates soon!

On the RET-positive aRMS side, this week should see the last experimental replicates of human cell lines being completed. The differences in growth rates of the human cell lines have been resolved, and from the data it seems as though the different initial cell populations does not effect the effect of the drug combinations. Analysis of the human cell line data will begin shortly and should conclude early next week.

 

05/10/2018 update

Dear GEAR Supporters,

Today I have updates on the two endotypes we are pursuing for the GEAR project.

On the Pax7:Foxo1 aRMS endotype sub-project, the cell line sent by our overseas collaborator did not recovered well. We are working to have a new cell line sent to us now. We will have cryopreserved cell sent to us late this month, which should be easy to recover in the lab.

On the RET-positive aRMS side, the final human experimental replicates have been completed. The data analysis for the human samples is now underway, and should be completed this week, alongside visualization of sample data. More to come one this shortly.

Thank you very much for your continued support!

 

05/17/2018 update

Dear GEAR Supporters,

Today I have updates on the two endotypes we are pursuing for the GEAR project.

I am working to set up a contract with World Courier to transfer cryopreserved cells from the Italian research group to ours. I will be working to set this up before next week, so we will have access to the PAX7:FOXO1 cell ine as soon as possible.

On the RET-positive aRMS side, the human and mouse data analysis is complete. Based on the results so far, we are taking a deeper look at the underlying change in biology caused by RET mutations in aRMS. The first step in this process is establishing aRMS cell lines that stably express RET. The lentivirus system arrived recently, and creating stable cell lines is my first goal in the next stage of this project.

Thank you very much for your continued support!